A Novel α – helix /β -sheet ratio as a Potential Metric For Studying Thermal Effects of Increased Formaldehyde Temperatures on Native Tissues

A Novel α – helix /β -sheet ratio as a Potential Metric For Studying Thermal Effects of Increased Formaldehyde Temperatures on Native Tissues | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Novel α – helix /β -sheet ratio as a Potential Metric For Studying Thermal Effects of Increased Formaldehyde Temperatures on Native Tissues Samuel Adeleke, Erioluwa Adesope, Oghenedoro Avbunufe, Christopher Igbeneghu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8773932/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Formaldehyde is a commonly used fixative in histological studies, with fixation outcomes significantly influenced by physical factors such as temperature. This study investigates the impact of varying fixation temperatures on tissue morphology and protein structural integrity using formaldehyde as the fixative. Ten (10) Wistar rats were sacrificed via cervical dislocation, and their liver, lungs, and kidneys were extracted. These organs were fixed in 10% Neutral Buffered Formalin at 25°C, 37°C, and 60°C for 24 hours. Standard tissue processing was followed, with 4 µm sections prepared for histological analysis using hematoxylin and eosin staining to assess tissue morphology. Additionally, 20 µm sections were analyzed using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy to examine protein spectral characteristics, employing the Agilent Cary 630 spectrometer in the 4000–600 cm⁻¹ IR range. Results revealed that tissues fixed at 25°C and 37°C showed superior preservation, while those at 60°C exhibited significant distortion for most of the tissues. Findings also showed that there was an elevation in beta-sheets as formalin temperature increases to 60°C compared with alpha-helixes on liver, lungs, and kidneys. However, performance test using receiver’s operating characteristics (ROC) curve showed potential of α – helix /β -sheet ratio to monitor effect of formalin-temperature increase on tissues and lung tissues appeared to be a perfect model for this study showing 100% accuracy, 100% sensitivity and 100% specificity to tell apart changes occuring to protein secondary structures between 25°C controlled formalin fixation and those at higher fixed at 37°C. These findings underscore the importance of optimizing fixation temperature to balance fixation speed and tissue integrity, drawing implications for antigen recovery in immunohistochemical studies. Formaldehyde Temperature Fixation Motifs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The crucial role of fixation in the realm of histology cannot be overemphasized. Fixation represents one of the principal stages in diagnostic pathology, serving a foundational role in pathological research. Its primary objective is to halt tissue autolysis and degradation, thereby enabling both anatomical and microscopic evaluation of tissue sections. The fixation process is integral to the extraction procedures, irrespective of the researcher's focus on lipids, proteins, RNA, or DNA [1,2]. Within the context of histopathology, tissue fixation is recognized as a fundamental and indispensable step. It alters the physicochemical attributes of tissues, thus modifying the reactivity of cellular components for staining purposes [3]. An optimal fixative should avert autolysis, decomposition, and putrefaction while simultaneously enhancing the structural solidity of tissue components for subsequent processing [4]. Extensive research on fixation chemistry has been documented since the early 20th century [4]. The practice of fixation has its origins in embalming, an ancient practice associated with religious observances in Egypt around 5000 BC. A pivotal milestone in the preservation of biological specimens occurred in 1868 with the introduction of formaldehyde into microscopic and gross anatomical protocols. Though discovered in Russia by Alexander Mikhaylovich Butlerov in 1859, its medical applications did not materialize until 1891 when Ferdinand Blum recognized its antibacterial and antimicrobial properties [5]. Formalin, however, emerges as the predominant fixative utilized in pathology laboratories worldwide, noted for its high precision, versatility, and user-friendly characteristics. The process of fixation involves diffusion of formalin, allowing it to penetrate deeply into the specimen. Effective penetration is essential for fixation; numerous physical variables influence the diffusion of formalin. The predominant factor that governs the fixation rate is the equilibrium shift favoring formaldehyde concentration. Additional determinants include temperature, pH, viscosity, and duration of fixation [4]. Missteps in tissue fixation can complicate rectification at later stages. Generally, adequate fixation time is contingent upon the tissue's size, consistency, and the type of fixative used. It is advisable that the volume of the fixative be roughly fifty times greater than that of the tissue itself. The main objective of fixation is to preserve tissues in a state as close to their original condition as possible, protecting them from adverse transformations such as autolysis and decomposition [6]. At room temperature, formaldehyde exists as a colorless gas with a potent odor. It is highly reactive, prone to polymerization, and flammable. The theoretical solubility of formaldehyde in water reaches 95% (w/w) at 120°C and is easily soluble in polar solvents, including water and alcohols. When in aqueous solution, formaldehyde hydrates to form methylene glycol. Methylene glycol molecules can react and combine to form polymers [4]. In aqueous solutions, formaldehyde rapidly hydrates into methylene glycol, resulting in an equilibrium that favors the hydrated form, such that true formaldehyde is present at less than 0.1%. The reactivity of formaldehyde solutions is recognized by chemists as a clock reaction, with the transformation of methylene glycol back to formaldehyde serving as an indicator of time progression [7]. Fixation is achieved via three primary mechanisms: covalent bonding, protein coagulation, and precipitate formation. Formaldehyde performs fixation through addition rather than coagulation, which occurs in two sequential phases; the initial rapid phase involves covalent bonding with amino acid amine groups (notably lysine, arginine, tyrosine, histidine, glutamine, and serine) within the first 12 hours. This is followed by a slower phase, during which methyl groups from glycol react to form methylene bridges [-CH2-] that link two protein molecules. In terms of nucleic acids, formaldehyde facilitates fixation through the action of methyl glycol groups that attach to free amine groups in the nucleic acid chains. Concurrently, lipoproteins are fixed via methyl glycol groups that arise from formalin hydrolysis, which bind to carbon particles or thiol groups [-SH]. This crosslinking mechanism is critical for the stabilization of proteins during fixation [7,8]. As a reactive electrophile, formaldehyde readily forms crosslinks with various functional groups of biological macromolecules [4]. When tissues are immersed in formalin, they are swiftly penetrated by methylene glycol and the smaller concentration of formaldehyde. The specific covalent reactions of the fixative with tissue depend on the consumption of formaldehyde as it forms bonds with tissue components, leading to an increase in formaldehyde concentration from the dissociation of methylene glycol as the reaction equilibrates [4]. Thus, the observed phenomenon where formaldehyde penetrates rapidly (as methylene glycol) yet fixes slowly (as carbonyl formaldehyde) is elucidated by the dynamic equilibrium between these two forms [9]. Several factors have been identified that can enhance the rate of fixation in routine histopathology [10]. However, parameters such as concentration extremes, inadequate fixation, over-fixation, and extended incubation periods can hinder epitope recovery during immunohistochemistry [10–13]. This fixation-induced alteration in protein conformation may lead to a loss of immunoreactivity. In scenarios necessitating elevated formalin temperatures for expedited processes, it is crucial to assess the implications on subsequent procedures. Variations in temperature and fixation duration typically yield samples exhibiting distinct degrees of cross-linking [14]. Notably, the specific influence of formalin temperature has been subject to investigation [14–16]. Although advocating for cold formalin fixation has been endorsed by certain studies [15–17], contrasting findings have emerged from other research groups [11]. Similarly, while Fox and colleagues suggested that increased temperature enhances formalin penetration, contemporary scholars criticize this practice, arguing that it leads to inadequately fixed tissue, particularly at the core, rendering it unsuitable for immunohistochemistry and other molecular analyses [15]. Routine fixation typically occurs at room temperature (20–25°C) for both histological and immunohistochemical applications, while histochemistry and electron microscopy processing may proceed at temperatures between 0 and 4°C [10, 18–19]. Lower temperature fixatives decelerate autolysis and diffusion of cellular components, whereas elevated temperatures accelerate chemical reactions involved in fixation. An increase in fixative temperature promotes rapid diffusion into the tissue and expedites the ensuing chemical reactions [9]. Some researchers report conflicting data on how temperature impacts tertiary protein structures [10, 20–22]. Nonetheless, there is a consensus that formalin temperatures exceeding 37–40°C can lead to unfavorable outcomes, where subtle alterations, not readily observable on Hematoxylin-Eosin-stained sections, can provoke protein misfolding at immunoreactive sites of the tissue [18, 23–24]. Despite this understanding, there exists a scarcity of literature examining protein conformational changes resulting from heated formalin fixation, as most research concerning heated formaldehyde's effects on secondary protein motifs (such as alpha-helices and beta-sheets) has predominantly utilized model proteins like bovine serum albumin, RNAse, hemoglobin, and ovalbumin, employing sophisticated methodologies including thermogravimetric analysis, differential scanning calorimetry, gel electrophoresis, transmission electron microscopy, and spectropolarimetry [20, 25–27]. Infrared (IR) spectroscopy is a long-established technique for analyzing the secondary structures of polypeptides and proteins. Advances in laser technology have facilitated the development of the Fourier transform (FT) method for IR data collection and precise digital subtraction. Modern computing has enabled rapid and substantial processing of FTIR data. The application of the technique can be expressed through its three modalities—transmission, transflection, and attenuated total reflectance (ATR)—which are employed based on the nature of the sample analyzed. Each approach presents distinct advantages and challenges depending on the specific context [28, 29]. When infrared radiation interacts with a sample, it engenders various vibrational modes. The extent of light absorption is intimately related to the molecular bonds present. Absorption frequencies are quantified as wavenumbers, typically ranging from 4000–600 cm − 1. The FTIR spectrum is expressed in terms of wavenumbers since this metric directly correlates with energy and frequency, thus simplifying spectrum interpretation. To prevent contamination peaks caused by air and water vapor, a background spectrum is recorded prior to sample analysis. The relationship between the background and the sample spectrum is essential for deriving the sample's absorption spectrum, which represents the various bond vibrations within the molecular structure [30]. The ATR modality of IR spectroscopy facilitates the analysis of water-based samples, which typically pose challenges for traditional FTIR methodologies due to their high IR absorption. The ATR-FTIR technique mitigates absorption issues by capturing molecular vibrations over a reduced path length across the sample. Through ATR mode configurations, molecular structural insights in terms of vibrational modes can be acquired from probing depths within micrometers via the evanescent wave generated at the interface between the ATR crystal and the sample medium [31]. FTIR instrumentation is fundamentally straightforward, comprising an infrared light source, such as a heated tungsten filament, which emits polychromatic light within the infrared range. This light is directed into a Michelson interferometer containing a beam splitter (e.g., germanium), dividing the beam into two components, one of which is stationary while the other oscillates with a defined frequency and amplitude. The two split beams recombine at the beam splitter, leading to interference based on optical path-length differences and subsequently directed to the sample [32]. Therefore, Fourier Transform Infrared (FTIR) spectroscopy has emerged as a pivotal technique for scrutinizing protein conformation in both aqueous and deuterated solutions, along with dried states. Its application has significantly proliferated in structural studies concerning protein secondary structures and dynamics over the previous decade. FTIR spectroscopy is a prominent method for structural characterization of proteins, peptides, and protein-membrane interactions. Although the technique lacks the capacity for atomic resolution of molecular structures, it is exceptionally sensitive to conformational alterations that occur during functional transitions or intermolecular interactions [32]. FTIR spectroscopy has proven invaluable for investigating the secondary structural composition, dynamic behaviors, conformational changes due to factors such as ligand binding, temperature fluctuations, pH variations, and pressure alterations, as well as overall protein stability and aggregation phenomena [28]. This study focuses on the effect of increasing formalin temperature on the secondary structures of proteins in histological tissues using α – helix /β -sheet ratio as marker to monitor transitions, highlighting potential implications of these changes for immunoreactivity and recovery of epitope sites during immunohistochemical applications. 2. Materials and methods 2.1 Animals Handling This research was conducted as an experimental study, receiving ethical clearance from the Bowen University Teaching Hospital (BUTH) in Ogbomosho, Oyo State, Nigeria. The study adhered to the National Institute of Health's guidelines regarding the Care and Use of Animals. It was executed within the Department of Medical Laboratory Science at Bowen University, Osun State, Nigeria. The animals utilized in this investigation were acquired and allowed to acclimatize in the Animal House facility at Bowen University for a duration of two weeks. A total of ten adult male Wistar rats, along with conventional rat feed and plastic cages with iron mesh, were employed throughout the study period of two months (June to August 2024). 2.2 Animal Sacrifice and Organ Processing The rats were administered a mild anesthetic and subsequently positioned on a flat surface. A swift downward force was applied while pulling the head upward to dislocate the cervical vertebrae, with death confirmed by the absence of respiration and heartbeat. Targeted organs, specifically the liver, lungs, and kidneys, were harvested for histological analysis. These organs were preserved using 10% neutral buffered formalin at different temperatures and underwent tissue processing. The fixation was conducted with 10% neutral buffered formalin at room temperature (25°C, group 1), at 37°C (group 2), and at 60°C (group 3) for a period of 24 hours, followed by dehydration through various grades of alcohol (50%, 70%, 80%, and 90%) and two changes of absolute alcohol sourced from Sigma-Aldrich, each lasting one hour [2]. Subsequently, the dehydrated tissues were cleared with two changes of toluene (Surgipath Medical Industries, Inc.), also for one hour each, and infiltrated with paraffin wax over three changes, each lasting one hour. The processed tissue sections were embedded in paraffin wax, and the resulting paraffin-embedded blocks were sectioned using a hand rotary microtome (ERM 3100 Hestion Histology Equipment). The sections were mounted onto glass slides (Unifrost microscope slide), then deparaffinized and rehydrated before being stained as outlined below. Stained sections were examined using an Olympus binocular light research microscope and recorded with a DXM1200 digital camera (Nikon). 2.3 Preparation of Slides Liver, lung, and kidney tissue sections that had been processed with paraffin wax were prepared for staining. Hematoxylin and Eosin (H and E) was the chosen staining protocol. All groups of slides underwent staining with Hematoxylin and Eosin. The slides were de-waxed in xylene for approximately five minutes and subsequently subjected to progressive grades of alcohol (100%, 90%, 70%, and 50%). Following a rinse in water, the sections were stained with Hematoxylin for around 15 minutes before rinsing again. A differentiation step using 1% acid alcohol for two seconds was performed, followed by another rinse in water. The sections were blued with tap water for ten minutes and stained with Eosin for 30 minutes. Excess staining was eliminated through dehydration with graded alcohol (50%, 70%, 90%, and 100%). The final stage involved clearing the slides in xylene and mounting them using DPX. The duplicates of the slides were analyzed utilizing an ATR-FTIR spectrometer. This spectrometer facilitated direct analysis of the sections, identifying similarities and discrepancies while assessing the biomolecules within the tissues that were either retained or lost, according to Adeleke et al [34]. 2.4 Fourier Transform Infrared (ATR-FTIR) Spectroscopy Methodology FTIR spectra of all the analyzed samples were obtained using the Agilent Cary 630 spectrometer from Agilent Technologies. The Attenuated Total Reflectance (ATR) technique utilized a diamond crystal for measurements taken across the IR spectrum from 4000 to 600 cm-1, employing 32 scans at a resolution of 16 cm-1. Each measurement was repeated five times. The resultant spectra underwent normalization, smoothing, and baseline corrections via Spectagyrph software [34]. 2.5 Statistical Analysis Data analysis of FTIR results was conducted employing IBM SPSS Statistics 25 software, with results presented in table format as mean ± standard deviation. An inferential statistic, specifically the t-test, was utilized for comparisons between two groups. Additionally, a Receiver Operating Characteristic (ROC) curve was employed to evaluate the performance, sensitivity, and specificity of the methodologies used in comparing temperature-controlled tissue groups. A significance level of P < 0.05 was established for the statistical analysis, with results also conveyed in a tabular format. 3. Results 3.1 Histological Results Figure 1a-c Shows photomicrograph of formalin-fixed Liver tissue in 25°C, 37°C, and 60°C respectively. Figure 2a-c Shows photomicrograph of formalin-fixed Lungs tissue in 25°C, 37°C, and 60°C respectively. Figure 3a-c Shows photomicrograph of formalin-fixed Kidney tissue in 25°C, 37°C, and 60°C respectively. Table 1 Peak assignment for alpha helix/Beta sheet and their relative spectral signal strength at different temperatures Peak Assignment Secondary protein structures References Signal strength Liver 25°C 37 °C 60°C 1654 α - helix 39, 40 Moderate Strong Weak 1625 β -sheet Moderate Strong Weak Lung 1654 α - helix 39, 40 Strong Weak Moderate 1625 β -sheet Strong Weak Moderate Kidney 1654 α - helix 39, 40 Weak Strong Moderate 1625 β -sheet Weak Strong Moderate Table 2 Comparing α-helix /β -sheet values of different organs at paired temperature ranges α – helix /β -sheet Ratio Organs Mean±SD 95%Confidence Interval Temperature range 25 ͦC 37 ͦC Mean difference Lower Upper t-value p-value Liver 1.02±0.001 0.79±0.0001 0.23 0.229 0.232 479.13 0.0001 Lung 1.03±0.002 1.14±0.001 -0.109 -0.111 -0.107 -130.08 0.000 Kidney 0.81±0.001 0.86±0.001 -0.0491 -0.051 -0.048 -84.62 0.001 25 ͦC 60 ͦC Liver 1.02±0.001 0.85±0.001 0.169 0.168 0.171 260.89 0.0001 Lung 1.03±0.002 0.92±0.001 0.11 0.108 0.112 116.41 0.001 Kidney 0.81±0.001 0.86±0.001 -0.049 -0.051 -0.048 -84.62 0.000 37 ͦC 60 ͦC Liver 0.79±0.0001 0.85±0.001 -0.061 -0.062 -0.06 -105.66 0.000 Lung 1.14±0.001 0.92±0.001 0.219 0.218 0.22 416.74 0.000 Kidney 0.86±0.001 0.86±0.001 -0.0004 -0.002 0.001 0.497 0.63 α – helix /β -sheet ratio showed statistical difference to distinguish tissues fixed at different temperature except for kidney at 37°C and 60°C Table 3 Performance of α – helix /β -sheet ratio as a metric of protein conformations on tissues formalin-fixed between 25°C and 37°C 95% CI p -value Tissues Temp Area SE Lower bound Upper bound Sensitivity Specificity Cut-off Liver 25 0.500 0.12 0.255 0.745 100% 50% 0.92 0.001* 37 0.00 0.00 0.00 0.001 71% 0% 0.79 0.00* Lung 25 #1.00 0.001 1.000 1.000 100% 100% 1.00 0.001* 37 #1.00 0.001 1.000 1.000 100% 100% 0.89 0.001* Kidney 25 0.462 0.138 0.191 0.733 100% 46% 0.83 0.78 37 0.001 0.000 0.001 0.001 100% 46% 0.86 0.78 Exploring the utility of α – helix /β -sheet ratio as metric of monitoring the effect of increasing formalin temperature (25°C- 37°C) on the structural integrity of proteins during fixation using AUC values, it was observed that only lung that showed potential as a experimental model to study this temperature impact, with α – helix /β -sheet showing excellent model performance (1.00), sensitivity (100%), specificity (100%) at statistical significance level (*) as shown in supplementary fig . 4 . #Excellent diagnostic performance. ROC, receiver operating characteristics; AUC, area under the curve; CI, confidence interval; Temp, Temperature; SE, standard error. Table 4 ROC Performance of α – helix /β -sheet ratio as a metric of protein conformations on tissues formalin-fixed between 25°C and 60°C 95% CI p -value Tissues Temp Area SE Lower bound Upper bound Sensitivity Specificity Cut-off Liver 25 0.500 0.134 0.238 0.762 100% 50% 0.92 1.00 60 0.000 0.000 0.000 0.000 83% 0% 0.85 0.01* Lung 25 #1.000 0.001 1.000 1.000 100% 100% 1.03 0.001* 60 #1.000 0.001 1.000 1.000 100% 100% 0.89 0.001* Kidney 25 0.00 0.00 0.000 0.001 71% 0% 0.81 0.01* 60 0.462 0.138 0.191 0.753 100% 46% 0.86 0.78 Exploring the utility of α – helix /β -sheet ratio as metric of monitoring the effect of increasing formalin temperature (25°C- 37°C) on the structural integrity of proteins during fixation using AUC values, it was observed that only lung that showed potential as a experimental model to study this with α – helix /β -sheet showing excellent model performance (1.00), sensitivity (100%), specificity (100%) at statistical significance level (*) as shown in supplementary fig . 5 . #Excellent performance. AUC, area under the curve; ROC, receiver operating characteristics; CI, confidence interval; Temp, Temperature; SE, standard error. Table 5 ROC Performance of α – helix /β -sheet ratio as a metric of protein conformations on tissues formalin-fixed between 37°C and 60°C 95% CI p -value Tissues Temp Area SE Lower bound Upper bound Liver 37 0.000 0.000 0.000 0.001 71% 0% 0.79 0.01* 60 0.000 0.000 0.000 0.001 85% 0% 0.85 0.01* Lung 37 #1.000 0.001 1.000 1.000 100% 100% 1.00 0.001* 60 #1.000 0.001 1.000 1.000 100% 100% 0.89 0.001* Kidney 37 0.467 0.129 0.214 0.719 100% 47% 0.86 0.80 60 0.467 0.129 0.214 0.719 100% 47% 0.86 0.83 Exploring the utility of α – helix /β -sheet ratio as metric of monitoring the effect of increasing formalin temperature (37°C- 60°C) on the structural integrity of proteins during fixation using AUC values, it was observed that only lung that showed potential as a experimental model to study this with α – helix /β -sheet showing excellent model performance (1.00), sensitivity (100%), specificity (100%) at statistical significance level (*) as shown in supplementary fig . 6 . #Excellent performance. ROC, receiver operating characteristics; AUC, area under the curve; CI, confidence interval; Temp, Temperature; SE, standard error. 4. Discussion Through the elevation of fixation temperature using standard formaldehyde fixative, this current research sought to examine how different temperature ranges affect both fixation effectiveness and the preservation of tissue architecture for histological examinations. The study aimed to evaluate how modifications in the temperature of the formaldehyde fixative influence specific histological specimens while exploring the associated morphological features, particularly regarding proteins that may either be preserved or transformed in response to heated fixatives. Tissue samples from the liver, lungs, and kidneys of Wistar rats were utilized for this investigation. After the organs were extracted, they were subjected to fixation in formaldehyde at temperatures of 25°C, 37°C, and 60°C, respectively. In the case of the liver tissue, hepatocytes were distinctly visible with minimal distortion at the 25°C setting. However, at 37°C, the fixation was inadequate, leading to uneven staining, particularly observed in the cellular centers [33]. Tissue architecture was notably preserved under the 25°C conditions, while at 60°C, the central vein was clearly outlined, though the cytoplasm of the liver cells was singularly observed with nuclei appearing shriveled (refer to Fig. 1 ). Observations for lung tissue indicated that air spaces underwent greater distortion under the 60°C condition compared to 37°C, suggesting that elevated heat induced denaturation, resulting in marked shrinkage and distortion associated with the lung structure. Nonetheless, the tissue matrix exhibited superior quality at 25°C compared to the higher temperatures, while staining qualities were distinctly differentiated at both 25°C and 37°C [35]. At 60°C, the central zones of the tissue were inadequately fixed, with significant loss of cellular and nuclear definition (as depicted in Fig. 2 ) [36]. In the kidney specimens, those fixed at 25°C displayed clear boundaries following staining, contrasting with the 37°C samples, which exhibited greater stain uptake but also maintained general architectural integrity. Conversely, at 60°C, noticeable tears and distortions were present, creating irregular spaces within the tissue architecture—an observation absents in samples fixed at 25°C and 37°C (as illustrated in Fig. 3 ) [35]. According to Bamisi and Alese, elevating the fixative temperature enhances the diffusion rate into the tissue, thus accelerating the reactive interactions between tissue and fixative. Nonetheless, extensive investigations into formaldehyde fixation mechanisms have established the concept of “rapid penetration, slow fixation” [36]. This observation implies that in the present study, the application of heat initiated the fixation process without providing sufficient penetration of the fixative. High temperatures were found to induce tissue fixation through thermal coagulation rather than enhancing the cross-linking process of formaldehyde [37]. The action mechanism of formaldehyde indicates that it penetrates swiftly (as methylene glycol) while the actual fixation is a slower process (as carbonyl formaldehyde) [9]. Therefore, the findings of this investigation propose that while heat facilitated a quicker reaction between tissue and fixative, it did not enhance the penetration rate. Overall, results indicate that while thermal conditions can accelerate fixation rates, they also correspondingly accelerate autolytic processes. It seems fixation proper only commenced once adequate penetration was achieved [3]. Additionally, the investigation into the effects of varying formalin temperatures on tissue specimens revealed a significant influence on the secondary structures of proteins, as evidenced by Fourier Transform Infrared Spectroscopy (FTIR) analyses. The predominant secondary structural motifs identified in proteins, namely alpha helices and beta sheets, are ubiquitous across various tissues in the body [38–39]. Research suggests that formalin fixation induces folding and denaturation, particularly affecting globular proteins characterized by alpha-helical structures due to the prevalent collagen found in soft tissues, while some proteins remain largely unaffected [10, 13, 20–21]. Some researchers argue that heat modifies the alpha-helix structures present in normal tissues into beta-sheet conformations, which are often indicative of tissue disorganization [10, 40–41]. However, the majority of studies examining this phenomenon have focused on the independent impacts of formalin and heat on protein models, with only a few studies evaluating their synergistic effects on proteins like RNAase A, bovine serum albumin, hemoglobin, and ovalbumin. Critical organs such as the liver, lungs, and kidneys are known for their extensive collagen fiber content. The liver, as the largest organ with its five lobes, plays vital roles in metabolism, transport, and detoxification. The kidneys are essential for homeostasis, facilitating ion transport and filtration functions, while the lungs enable gas exchange necessary for respiration [43]. These organs predominantly comprise globular proteins, predominantly alpha-helices interlaced with beta-sheets, which are essential for their various physiological functions. In supplementary Figs. 1 and 2, the observed shift in protein bands corresponding to Amide I (1625 cm-1) and Amide II (1520 cm-1) towards beta-sheet conformations provides evidence of temperature's impact on protein structure. Notably, more pronounced peaks were found at 25°C and 37°C in comparison to a decrease in intensity at 60°C. Specifically, liver and lung samples fixed at 60°C displayed weaker absorbance compared to those treated at 25°C and 37°C, indicating potential unfolding and dissociation associated with the loss of intermolecular hydrogen bonds within the beta-sheet structures. Interestingly, the lung tissue fixed at 25°C showed more intense protein peaks than tissue fixed at 37°C, aligning with previous studies that indicated a greater binding rate of formalin at 37°C versus 25°C. This leads to the hypothesis of ongoing protein conformational changes occurring at lower temperatures rather than at elevated temperatures like 60°C [37]. The weak intensity of beta-sheets observed at 60°C suggests potential conformational alterations in proteins, with some scholars attributing this to heat coagulation rather than formalin fixation [37]. While the overall implications of this hypothesis have yet to be thoroughly investigated, the present findings revealed a marked reduction in peak intensity for lung tissues fixed at 60°C compared to those fixed at 37°C, indicating possible protein structural distortion. This phenomenon may be attributed to the activation of cellular enzymes at elevated temperatures, which can lead to cellular degradation in enzyme-rich tissues such as the liver [15–16]. These observations are consistent with similar research advocating for formalin fixation at higher temperatures but over shorter durations [37]. Conversely, the kidneys fixed in formalin at 60°C exhibited stronger protein peaks at Amide I (1630 cm-1) and Amide II (1530 cm-1), corresponding to beta-sheet structures, followed by those fixed at 25°C, which also outperformed 37°C fixed kidney tissues in peak intensity. Evidence from kidney samples indicates significant protein conformational changes occurring at 60°C, as reflected by the pronounced beta-sheet spectra, suggesting enhanced stability of internal molecular bonds necessary for subsequent tissue processing compared to 25°C and 37°C fixed samples (Table 1 , Fig. 6 a-c). The observed effects of temperature on protein structure appear inconsistent; while higher temperatures promote tissue stability in the kidneys, detrimental outcomes of heat on the fixation processes, particularly noted in liver and lung tissues, have also been documented [15]. This study suggests that fixation at elevated temperatures may adversely affect liver and lung tissues more than those at room temperature, potentially due to activation of cellular enzymes at elevated temperatures leading to improper fixation prior to spectral analysis. The spectral data for the liver indicates that both Amide I and Amide II peaks corresponding to beta sheets reflect a tissue-formalin interaction, albeit subtly as shown in supplementary Fig. 1–3. This finding contrasts with reports asserting that formalin penetrates liver tissue slowly [15]. Consequently, fixation at higher temperatures likely compromises native protein retention and is unnecessary for effectively preserving cell-to-cell relationships within the tissue matrix [15]. Again, in this study, we examined the relative abundance of native alpha-helices compared to beta-sheets in proteins at 25°C as the temperature increases across liver, lung, and kidney tissues. The ambient temperature of 25°C is frequently cited as optimal for most routine histological analyses [22, 44]. Our results indicated that at this temperature, alpha-helices were predominantly present in liver and lung tissues, while in the kidney, beta-sheets were more prevalent, as illustrated in Table 2 and Figs. 4 – 6 . The underlying factors contributing to the elevated beta-sheet presence in kidney tissues remain somewhat ambiguous, but it is likely influenced by formalin-induced conformations. This suggests that the conversion rates from alpha-helix to beta-sheet in lung and liver tissues are relatively stable at room temperature when compared to kidney tissues. Nonetheless, both liver and kidney tissues exhibited a decrease in alpha-helix content relative to beta-sheets at higher temperatures (37°C and 60°C), indicating the significant role of elevated temperatures in progressive entropy leading to protein denaturation [18, 23–24, 41, 45]. This observation aligns with multiple studies that have pointed to heat as a factor in the transition from alpha-helices to beta-sheets. The increase in beta-sheets has been linked to both direct and indirect transformations originating from the unfolding of alpha-helices and random coils to anti-parallel beta-sheets [27, 40, 46]. Conversely, lung tissue maintained a relatively stable alpha-helix content at 37°C compared to the beta-sheet content. This phenomenon may suggest that 37°C presents a limiting condition for the conversion of alpha-helices to beta-sheets while simultaneously promoting the conversion of unordered random coil structures to alpha-helices [27]. The overarching conclusion of this research indicates that with the progressive increase in formalin temperature from 37°C to 60°C, the beta-sheet content becomes more pronounced compared to alpha-helices, highlighting the notion of heat-induced changes in secondary structure motifs observed across all organs [45]. The basis for these alterations is likely due to the synergistic and interactive influences of formalin and heat on the histological tissues, leading to protein unfolding via the destabilization of hydrophilic bonds within the tissue matrix [22, 42, 44, 47]. Importantly, the increase in formalin temperature from 25°C to 37°C and 60°C had minimal effect on the protein substructures of compact tissues like liver and kidney, as measured by the alpha-helix/beta-sheet ratio, which assessed the sensitivity of these tissues to temperature fluctuations. The relative statistical analyses indicated a greater presence of alpha-helices at 25°C in comparison to 60°C, where beta-sheets predominated (Table 2 ). This finding implies conformational changes wherein normal tissues, under specific favorable conditions, transition to a more destabilized state in pathological scenarios induced naturally or artificially [38, 48]. The cut-off points also suggest higher values at 25°C compared with values at 37°C and 60°C is key to identifying realistic effects especially on liver and lungs. However, only lung tissues exhibited significant changes, demonstrating a measurable impact of fixation temperature on protein secondary structures, as the calculated ratios revealed not only statistical significance but also superior performance across tissues fixed at varying temperatures (Tables 3 – 4 ). This aligns with previous findings where the same ratio was effectively used to investigate protein conformational changes in breast cancer with remarkable accuracy, achieving sensitivity and specificity above 90% [38, 49]. The pronounced effect of formalin temperature on lung tissues, as indicated by this innovative metric, arises from its more loosely arranged parenchyma containing air spaces. The connective tissues in the lungs are thinner and more elastic compared to the denser, fibrous structures of the kidney and liver, which are optimized for metabolic and filtration functions needing a compact surface area [43]. Consequently, the diffusion and penetration of formalin within the lung cavities is enhanced by elevated temperature, leading to corresponding alterations in protein structures, a phenomenon that was less pronounced in the other tissues evaluated for temperature-related protein changes. 4.1 Potential Consequences of Heat-Induced Conversion from α-Helix to β-Sheet for Epitope Retrieval Epitope retrieval is a critical procedure for formalin-fixed and cross-linked tissues, employing heat and enzymatic techniques [50]. This process is based on the principle that the cross-linking of surface proteins can be reversed under specific conditions, including temperature, pH, concentration, and buffer composition [51]. Notably, while formalin alters certain surface proteins, it can also leave some regions untouched, resulting in partial fixation or peripheral fixation [10, 21, 24, 50]. The varying degrees of disruption to tertiary protein structures have been associated with factors like solvent polarity, pH, concentration, duration, and temperature [13, 21, 27]. Traditionally, the optimal temperature for most histological techniques ranges from 20–25°C [18]. Within this temperature range, the preservation of both secondary and tertiary protein conformations during formalin fixation is typically reversible through appropriate interventions. Numerous studies have investigated the effects of 4°C and 25°C in histological, histochemical, and immunohistochemical contexts [10, 19, 23]. However, temperatures above 40°C can compromise tissue integrity despite enhancing penetration. For example, formalin at 40°C tends to fix tissues peripherally, leaving the central regions largely unaffected, and this phenomenon has been correlated with diminished immunoreactivity [23]. Consequently, conditions that impair or weaken immunoreactive sites on tissue diminish the likelihood of successful epitope recognition by antibodies [12, 24]. Nonetheless, the simultaneous effects of heat and formalin on secondary protein structures and their implications for epitope retrieval and antibody recognition in immunohistochemistry remain incompletely understood. Thus, this section endeavors to conceptually elucidate the mechanisms behind these temperature-induced protein alterations and their unintended consequences on antigen retrieval and antibody binding. Formalin primarily interacts with lysine and cysteine residues, leading to the formation of methylol groups, which subsequently react with other nucleophiles such as tyrosine, glutamine, histidine, arginine, and tryptophan [20, 50]. These methylol groups, also referred to as methylene bridges, are integral to the structural integrity of tissues [22]. A range of studies has explored the impact of heated formalin on non-native protein models [20, 24, 25–26, 44]. Typically, N-terminal amino acid residues are less reactive with formalin, while side chains of amino acids like cysteine, histidine, lysine, and arginine demonstrate higher reactivity in native protein contexts [10, 50]. Lysine and cysteine, being the most accessible, are likely to undergo significant conformational changes, forming intermolecular crosslinks in response to formaldehyde [10, 26, 51–52]. The diverse reactivity of these exposed groups on protein surfaces is crucial for epitope recovery and antibody recognition [10, 13, 52]. The retrieval of thermally stabilized crosslinks, or antigen retrieval, can be achieved through heating at 65°C and enzymatic treatments [10, 26]. The ability to revert cross-linked proteins to their native forms under normal conditions indicates no irreversible destruction has occurred. Adhering to the prevailing consensus that increased temperatures induce conformational shifts from alpha-helix to beta-sheet structures in proteins—due to destabilization of critical hydrogen bonds—it can be posited that alpha-helices, commonly found in amino acids such as lysine within native proteins, retain their structural and cross-linking associations even after formalin treatment, allowing for their reversion when favorable conditions are met [27, 40, 44, 46]. Nevertheless, observations from this study suggest that elevated temperatures might prompt irreversible conversions of alpha-helical amino acids to beta-sheet structures, particularly in long-chain poly-L-lysine native tissues, regardless of optimal conditions and retrieval methods [27]. This heat-induced transformation is attributed to the gauche isomerization of CH2 groups in the hydrocarbon side chains of lysine amino acids within both reactive and non-reactive tissue domains [27, 41, 50]. Increased temperatures have been shown to accelerate the conversion of alpha-helices to beta-sheets, the latter known for their comparatively weakened exposed intermolecular hydrogen bonds, resulting in enhanced thermal stability but reduced digestibility [40, 42, 44]. Consequently, this alteration renders tissues less susceptible to subsequent interactions, such as antibody adherence. Alpha-helices demonstrate more local mobility within their native pools compared to the relatively rigid beta-strands, which typically require additional energy for unfolding. This phenomenon may lead to a transient-post permanent loss of essential immunoreactive sites necessary for antibody binding, leaving only protein components unaffected by formalin or heat to interact with antibodies, often resulting in weak or absent staining visibility [20, 24, 50]. Additionally, amino acids that are known to exhibit beta-sheet conformations, such as isoleucine, tyrosine, tryptophan, and valine, are secondarily influenced by formalin, lending support to the hypothesis that the transformation from alpha-helix to a fundamentally different conformation, such as beta-sheet, alters protein surfaces, revealing amino acids that are not characteristic of the native protein but emerge as byproducts of heated formalin interactions affecting protein structural integrity [53]. 5. Conclusion While various methods, including thermogram and differential scanning calorimetry and circular dichroism, have been employed, they are often considered less sensitive to minor transitions in protein denaturation at the secondary structure level [25]. However, Fourier-transform infrared (FTIR) spectroscopy not only captured the qualitative and quantitative changes induced by increased formalin temperature relative to the standard 25°C, but the derivative values from the alpha-helix/beta-sheet ratio subjected to statistical analysis revealed that it could be utilized to detect changes in protein conformation as temperature rises, which may affect downstream applications like immunohistochemistry. This research indicates that Fourier transform infrared spectroscopy (FTIR) can provide a comprehensive method for examining the impact of thermal exposure on proteins, applicable at various stages of laboratory processes, particularly during the analytical phase of tissue preparation, which encompasses fundamental procedures like tissue fixation. The ratio of α-helix to β-sheet structures has demonstrated considerable sensitivity in identifying changes at the protein level, especially in loosely organized tissues such as lung tissue, with a commendable degree of precision. This advancement signals the potential incorporation into quality management systems within the histopathology domain, promising significant improvements in the reliability and validity of real-time histopathological diagnoses. 6 Future Directions While it is believed that rat anatomical characteristics approximates that of human, future studies on real human samples would be commendable. It is also advisable for forthcoming research to utilize the α-helix to β-sheet ratio as a parameter to assess the thermal effects utilizing a larger sample size. Additionally, examining temperature elevations beyond those studied here in solid organs may prove beneficial in evaluating the efficacy of this innovative marker in various contexts. While the lung has been established as an excellent model for investigating the consequences of high-temperature formalin fixation, other tissue types should be considered in future studies to broaden the scope of this research. The use of image analysis and artificial intelligence might also bring new insights into patterns of formalin-temperature-induced changes on organs opening up a roadmap to empirical quality management system in histopathological laboratories. Declarations Funding: The authors did not receive any research grant or fund from any place or organization for the completion of this present study Author Contribution Authors contributions AST: Conceptualization, Supervision, Data analysis and interpretation, Manuscript writing and editing; AEA; Data collection, first draft writing; AOG: Data collection, Manuscript review; IC: Manuscript revision Data Availability All necessary data supporting our findings for this present study are enclosed within the manuscript References Howat WJ, and Wilson BA. (2014). Tissue fixation and the effect of molecular fixatives on downstream staining procedures. Methods , 70 (1): 12–19. https://doi.org/10.1016/j.ymeth.2014.01.022 Oluwaloye GT, Jenakevwe OF, Onyegbula KC, Bankole JK. and Seyi Samson Enitan. (2023). Assessment of different formalin concentrations on the histochemical staining of liver, kidneys and lungs of Wistar rats. 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(2013). Conformational distribution and α-helix to β-sheet transition of human amylin fragment dimer. Biomacromolecules , 15 (1): 122-131. Litvinov RI, Faizullin DA, Zuev YF. and Weisel JW. (2012). The -helix to β-sheet transition in stretched and compressed hydrated fibrin clots. Biophys J. 103(5): 1020-1027. Abrusan G, Marsh JA. (2016). Alpha helices are more robust to mutations than Beta strands. PLoS Computational Biology, 12 (12): e1005242 Ghimire H, Garlapati C, Janssen EAM, Krishnamurti U, Qin G, Aneja R, et al (2020). Protein Conformational Changes in Breast Cancer Sera Using Infrared Spectroscopic Analysis. Cancers (Basel), 12 (7):1708. doi: 10.3390/cancers12071708. PMID: 32605072. Sompuram SR, Vani K, Messana E, Bogen SA. (2002). A molecular mechanism of formalin fixation and antigen retrieval. American Journal of Clinical Pathology , 121 (2): 190-199 Tayri-Wilk T, Slavin M, Zamel J, Blass A, Cohen S, Motzik A, Sun X, Shalev DE, Ram O. and Kalisman N. (2020). Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins. Nature Communications , 11 :3128 Vani K, Bogen SA, Sompuram SR. (2006). A high throughput combinatorial library technique for identifying formalin-sensitive epitopes. J Immunol Methods , 317 (1-2):80-89. Liu Y, Liu R, Mou Y, Zhou G. (2011). Spectroscopic identification of interactions of formaldehyde with bovine serum albumin. J. Biochem Mol. Toxicol 25 (2): 95-100. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfig.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8773932","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588575477,"identity":"b7154dd3-bc0f-4939-9565-976bdb937ef7","order_by":0,"name":"Samuel Adeleke","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABJklEQVRIiWNgGAWjYNCCAiA+wNzA2MBgkwAVYiagxQCkhRGkJY10LYcJa5FvP50m8cGAQZ7veGPjwxkV5/P4JZKPPWCosE5skG6/gNX8M7nbJGcYMBjOPHOw2XDDmdvFkjPS0g0YzqQnNsicKcDupNxt0jwGDIwbbiS2ST5su5244XaOmQRj2+HEBomcBGxa5PvfbpP+Y8Bgv+H+w/afD/+dA2rJ/ybB+A+3FoYbQFuAdiVuuMHYxrix4QDIFjYJxgaQlvQDWB124+1myx4DieSZZxKbJWccSy6WnP/MTCLhWLpxm0QO9hDrz91440eFjW3f8cMHP/bU2OXx8xx+JvGhxlq2XyL9AVY9ECCBxgd5go2BxwCPFuyAHZ8to2AUjIJRMHIAABQlbK2ES0umAAAAAElFTkSuQmCC","orcid":"","institution":"Bowen University","correspondingAuthor":true,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Adeleke","suffix":""},{"id":588575478,"identity":"05a96662-7cdf-402c-8a8a-68a1dab1ce5a","order_by":1,"name":"Erioluwa Adesope","email":"","orcid":"","institution":"Bowen University","correspondingAuthor":false,"prefix":"","firstName":"Erioluwa","middleName":"","lastName":"Adesope","suffix":""},{"id":588575479,"identity":"b92a6b13-fbe3-4ec2-8776-5a9d64955cb5","order_by":2,"name":"Oghenedoro Avbunufe","email":"","orcid":"","institution":"Bowen University","correspondingAuthor":false,"prefix":"","firstName":"Oghenedoro","middleName":"","lastName":"Avbunufe","suffix":""},{"id":588575481,"identity":"c569df2f-fd92-448d-9449-2db5a70bf2d0","order_by":3,"name":"Christopher Igbeneghu","email":"","orcid":"","institution":"Ladoke Akintola University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"Igbeneghu","suffix":""}],"badges":[],"createdAt":"2026-02-03 09:53:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8773932/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8773932/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102585017,"identity":"c36403a8-5782-4980-8759-d85f32cf9dbf","added_by":"auto","created_at":"2026-02-13 10:07:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":774900,"visible":true,"origin":"","legend":"\u003cp\u003eThe Liver tissue: (a) Fixed in formalin under 25°C condition; portal vein, clearly observable, hepatocytes are also clearly differentiated, with an adequate staining intensity, H\u0026amp;E. (b) Fixed in formalin under 37°C condition; the hepatocytes appear slightly distorted with nucleus appearing shrunk; the center of the tissue also appears under-fixed, H\u0026amp;E. (c) Fixed in formalin under 60°C condition; central vein clearly observed. The liver cells appearing singly, however nucleus appears shrunk. Staining is adequate, H\u0026amp;E (×400).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8773932/v1/a25ab2e35a3fa60f5fad6020.png"},{"id":102585016,"identity":"072229b2-6e9d-4ca1-b5c1-eb6d77706be2","added_by":"auto","created_at":"2026-02-13 10:07:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":682859,"visible":true,"origin":"","legend":"\u003cp\u003eThe lung tissue: (a) Fixed in formalin under 25°C condition; air spaces clearly seen; regular ratio between air space and blood stream, alveolar space with normal cellular components, H\u0026amp;E. (b) Fixed in formalin under 37°C condition; air spaces are enlarged, lung architecture appears stretched, H\u0026amp;E. (c) Fixed in formalin under 60°C condition; meshwork cannot be clearly differentiated, lung tissue appears shrunk, staining appears eosinophilic, H\u0026amp;E (×400).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8773932/v1/22208aa213df61404a9e7ed9.png"},{"id":102585021,"identity":"b9731247-6b1d-4b4b-bc28-cbe8b3c37ed8","added_by":"auto","created_at":"2026-02-13 10:07:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":689905,"visible":true,"origin":"","legend":"\u003cp\u003eThe kidney tissue: (a) Fixed in formalin under 25°C condition; staining is adequate with clear nucleocytoplasmic staining, H\u0026amp;E. (b) Fixed in formalin under 37°C condition; nucleocytoplasmic detail adequate, but not clearly distinguished. Staining is more intense at the edge of tissue, H\u0026amp;E. (c) Fixed in formalin under 60°C condition; nucleus appears shrunk, staining is less intense and weak, and there is less distinct cellular borders H\u0026amp;E (×400).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8773932/v1/53a2073995d9d4000aa28b6b.png"},{"id":103049328,"identity":"0ac41ad9-1f69-44f2-992d-f363e0d26ca2","added_by":"auto","created_at":"2026-02-20 07:39:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a-c): Line plot of alpha-helix and beta-sheet proportion on liver tissue fixed at 25 ͦ C, 37 ͦ C and 60 ͦ C respectively\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8773932/v1/db170e064309b9e2f4aed722.png"},{"id":102585015,"identity":"af2db4cf-af9e-4a2c-992f-2c0ef802f079","added_by":"auto","created_at":"2026-02-13 10:07:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":35065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a-c): Line plot of alpha-helix and beta-sheet proportion on lung fixed at 25 ͦ C, 37 ͦ C and 60 ͦ C respectively\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8773932/v1/4d0becd86143b86bd3d89421.png"},{"id":102585019,"identity":"17e17b19-454e-442b-8f2a-5cd3a7c115f5","added_by":"auto","created_at":"2026-02-13 10:07:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":33847,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a-c): Line plot of alpha-helix and beta-sheet proportion on kidney fixed at 25 ͦ C, 37 ͦ C and 60 ͦ C respectively\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8773932/v1/64ebbab5a0d0961c2cd5d0f0.png"},{"id":103654973,"identity":"a157ce03-58a9-48ae-9b48-4b4184419ccc","added_by":"auto","created_at":"2026-02-28 14:25:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3325609,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8773932/v1/b5970176-fc1d-4d78-a925-2b98551caac2.pdf"},{"id":102585020,"identity":"9898fd76-c365-4c6d-8b73-4e17f3bbcdc9","added_by":"auto","created_at":"2026-02-13 10:07:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":174077,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfig.docx","url":"https://assets-eu.researchsquare.com/files/rs-8773932/v1/14f539ae968d7bcc37be6b8f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Novel α – helix /β -sheet ratio as a Potential Metric For Studying Thermal Effects of Increased Formaldehyde Temperatures on Native Tissues","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe crucial role of fixation in the realm of histology cannot be overemphasized. Fixation represents one of the principal stages in diagnostic pathology, serving a foundational role in pathological research. Its primary objective is to halt tissue autolysis and degradation, thereby enabling both anatomical and microscopic evaluation of tissue sections. The fixation process is integral to the extraction procedures, irrespective of the researcher's focus on lipids, proteins, RNA, or DNA [1,2]. Within the context of histopathology, tissue fixation is recognized as a fundamental and indispensable step. It alters the physicochemical attributes of tissues, thus modifying the reactivity of cellular components for staining purposes [3]. An optimal fixative should avert autolysis, decomposition, and putrefaction while simultaneously enhancing the structural solidity of tissue components for subsequent processing [4].\u003c/p\u003e \u003cp\u003eExtensive research on fixation chemistry has been documented since the early 20th century [4]. The practice of fixation has its origins in embalming, an ancient practice associated with religious observances in Egypt around 5000 BC. A pivotal milestone in the preservation of biological specimens occurred in 1868 with the introduction of formaldehyde into microscopic and gross anatomical protocols. Though discovered in Russia by Alexander Mikhaylovich Butlerov in 1859, its medical applications did not materialize until 1891 when Ferdinand Blum recognized its antibacterial and antimicrobial properties [5].\u003c/p\u003e \u003cp\u003eFormalin, however, emerges as the predominant fixative utilized in pathology laboratories worldwide, noted for its high precision, versatility, and user-friendly characteristics. The process of fixation involves diffusion of formalin, allowing it to penetrate deeply into the specimen. Effective penetration is essential for fixation; numerous physical variables influence the diffusion of formalin. The predominant factor that governs the fixation rate is the equilibrium shift favoring formaldehyde concentration. Additional determinants include temperature, pH, viscosity, and duration of fixation [4]. Missteps in tissue fixation can complicate rectification at later stages. Generally, adequate fixation time is contingent upon the tissue's size, consistency, and the type of fixative used. It is advisable that the volume of the fixative be roughly fifty times greater than that of the tissue itself. The main objective of fixation is to preserve tissues in a state as close to their original condition as possible, protecting them from adverse transformations such as autolysis and decomposition [6].\u003c/p\u003e \u003cp\u003eAt room temperature, formaldehyde exists as a colorless gas with a potent odor. It is highly reactive, prone to polymerization, and flammable. The theoretical solubility of formaldehyde in water reaches 95% (w/w) at 120\u0026deg;C and is easily soluble in polar solvents, including water and alcohols. When in aqueous solution, formaldehyde hydrates to form methylene glycol. Methylene glycol molecules can react and combine to form polymers [4]. In aqueous solutions, formaldehyde rapidly hydrates into methylene glycol, resulting in an equilibrium that favors the hydrated form, such that true formaldehyde is present at less than 0.1%. The reactivity of formaldehyde solutions is recognized by chemists as a clock reaction, with the transformation of methylene glycol back to formaldehyde serving as an indicator of time progression [7].\u003c/p\u003e \u003cp\u003eFixation is achieved via three primary mechanisms: covalent bonding, protein coagulation, and precipitate formation. Formaldehyde performs fixation through addition rather than coagulation, which occurs in two sequential phases; the initial rapid phase involves covalent bonding with amino acid amine groups (notably lysine, arginine, tyrosine, histidine, glutamine, and serine) within the first 12 hours. This is followed by a slower phase, during which methyl groups from glycol react to form methylene bridges [-CH2-] that link two protein molecules. In terms of nucleic acids, formaldehyde facilitates fixation through the action of methyl glycol groups that attach to free amine groups in the nucleic acid chains. Concurrently, lipoproteins are fixed via methyl glycol groups that arise from formalin hydrolysis, which bind to carbon particles or thiol groups [-SH]. This crosslinking mechanism is critical for the stabilization of proteins during fixation [7,8]. As a reactive electrophile, formaldehyde readily forms crosslinks with various functional groups of biological macromolecules [4]. When tissues are immersed in formalin, they are swiftly penetrated by methylene glycol and the smaller concentration of formaldehyde. The specific covalent reactions of the fixative with tissue depend on the consumption of formaldehyde as it forms bonds with tissue components, leading to an increase in formaldehyde concentration from the dissociation of methylene glycol as the reaction equilibrates [4]. Thus, the observed phenomenon where formaldehyde penetrates rapidly (as methylene glycol) yet fixes slowly (as carbonyl formaldehyde) is elucidated by the dynamic equilibrium between these two forms [9].\u003c/p\u003e \u003cp\u003eSeveral factors have been identified that can enhance the rate of fixation in routine histopathology [10]. However, parameters such as concentration extremes, inadequate fixation, over-fixation, and extended incubation periods can hinder epitope recovery during immunohistochemistry [10\u0026ndash;13]. This fixation-induced alteration in protein conformation may lead to a loss of immunoreactivity. In scenarios necessitating elevated formalin temperatures for expedited processes, it is crucial to assess the implications on subsequent procedures. Variations in temperature and fixation duration typically yield samples exhibiting distinct degrees of cross-linking [14]. Notably, the specific influence of formalin temperature has been subject to investigation [14\u0026ndash;16]. Although advocating for cold formalin fixation has been endorsed by certain studies [15\u0026ndash;17], contrasting findings have emerged from other research groups [11]. Similarly, while Fox and colleagues suggested that increased temperature enhances formalin penetration, contemporary scholars criticize this practice, arguing that it leads to inadequately fixed tissue, particularly at the core, rendering it unsuitable for immunohistochemistry and other molecular analyses [15]. Routine fixation typically occurs at room temperature (20\u0026ndash;25\u0026deg;C) for both histological and immunohistochemical applications, while histochemistry and electron microscopy processing may proceed at temperatures between 0 and 4\u0026deg;C [10, 18\u0026ndash;19]. Lower temperature fixatives decelerate autolysis and diffusion of cellular components, whereas elevated temperatures accelerate chemical reactions involved in fixation. An increase in fixative temperature promotes rapid diffusion into the tissue and expedites the ensuing chemical reactions [9]. Some researchers report conflicting data on how temperature impacts tertiary protein structures [10, 20\u0026ndash;22]. Nonetheless, there is a consensus that formalin temperatures exceeding 37\u0026ndash;40\u0026deg;C can lead to unfavorable outcomes, where subtle alterations, not readily observable on Hematoxylin-Eosin-stained sections, can provoke protein misfolding at immunoreactive sites of the tissue [18, 23\u0026ndash;24]. Despite this understanding, there exists a scarcity of literature examining protein conformational changes resulting from heated formalin fixation, as most research concerning heated formaldehyde's effects on secondary protein motifs (such as alpha-helices and beta-sheets) has predominantly utilized model proteins like bovine serum albumin, RNAse, hemoglobin, and ovalbumin, employing sophisticated methodologies including thermogravimetric analysis, differential scanning calorimetry, gel electrophoresis, transmission electron microscopy, and spectropolarimetry [20, 25\u0026ndash;27].\u003c/p\u003e \u003cp\u003eInfrared (IR) spectroscopy is a long-established technique for analyzing the secondary structures of polypeptides and proteins. Advances in laser technology have facilitated the development of the Fourier transform (FT) method for IR data collection and precise digital subtraction. Modern computing has enabled rapid and substantial processing of FTIR data. The application of the technique can be expressed through its three modalities\u0026mdash;transmission, transflection, and attenuated total reflectance (ATR)\u0026mdash;which are employed based on the nature of the sample analyzed. Each approach presents distinct advantages and challenges depending on the specific context [28, 29].\u003c/p\u003e \u003cp\u003eWhen infrared radiation interacts with a sample, it engenders various vibrational modes. The extent of light absorption is intimately related to the molecular bonds present. Absorption frequencies are quantified as wavenumbers, typically ranging from 4000\u0026ndash;600 cm\u0026thinsp;\u0026minus;\u0026thinsp;1. The FTIR spectrum is expressed in terms of wavenumbers since this metric directly correlates with energy and frequency, thus simplifying spectrum interpretation. To prevent contamination peaks caused by air and water vapor, a background spectrum is recorded prior to sample analysis. The relationship between the background and the sample spectrum is essential for deriving the sample's absorption spectrum, which represents the various bond vibrations within the molecular structure [30]. The ATR modality of IR spectroscopy facilitates the analysis of water-based samples, which typically pose challenges for traditional FTIR methodologies due to their high IR absorption. The ATR-FTIR technique mitigates absorption issues by capturing molecular vibrations over a reduced path length across the sample. Through ATR mode configurations, molecular structural insights in terms of vibrational modes can be acquired from probing depths within micrometers via the evanescent wave generated at the interface between the ATR crystal and the sample medium [31]. FTIR instrumentation is fundamentally straightforward, comprising an infrared light source, such as a heated tungsten filament, which emits polychromatic light within the infrared range. This light is directed into a Michelson interferometer containing a beam splitter (e.g., germanium), dividing the beam into two components, one of which is stationary while the other oscillates with a defined frequency and amplitude. The two split beams recombine at the beam splitter, leading to interference based on optical path-length differences and subsequently directed to the sample [32].\u003c/p\u003e \u003cp\u003eTherefore, Fourier Transform Infrared (FTIR) spectroscopy has emerged as a pivotal technique for scrutinizing protein conformation in both aqueous and deuterated solutions, along with dried states. Its application has significantly proliferated in structural studies concerning protein secondary structures and dynamics over the previous decade. FTIR spectroscopy is a prominent method for structural characterization of proteins, peptides, and protein-membrane interactions. Although the technique lacks the capacity for atomic resolution of molecular structures, it is exceptionally sensitive to conformational alterations that occur during functional transitions or intermolecular interactions [32]. FTIR spectroscopy has proven invaluable for investigating the secondary structural composition, dynamic behaviors, conformational changes due to factors such as ligand binding, temperature fluctuations, pH variations, and pressure alterations, as well as overall protein stability and aggregation phenomena [28].\u003c/p\u003e \u003cp\u003eThis study focuses on the effect of increasing formalin temperature on the secondary structures of proteins in histological tissues using α \u0026ndash; helix /β -sheet ratio as marker to monitor transitions, highlighting potential implications of these changes for immunoreactivity and recovery of epitope sites during immunohistochemical applications.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals Handling\u003c/h2\u003e \u003cp\u003e This research was conducted as an experimental study, receiving ethical clearance from the Bowen University Teaching Hospital (BUTH) in Ogbomosho, Oyo State, Nigeria. The study adhered to the National Institute of Health's guidelines regarding the Care and Use of Animals. It was executed within the Department of Medical Laboratory Science at Bowen University, Osun State, Nigeria. The animals utilized in this investigation were acquired and allowed to acclimatize in the Animal House facility at Bowen University for a duration of two weeks. A total of ten adult male Wistar rats, along with conventional rat feed and plastic cages with iron mesh, were employed throughout the study period of two months (June to August 2024).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Animal Sacrifice and Organ Processing\u003c/h2\u003e \u003cp\u003eThe rats were administered a mild anesthetic and subsequently positioned on a flat surface. A swift downward force was applied while pulling the head upward to dislocate the cervical vertebrae, with death confirmed by the absence of respiration and heartbeat. Targeted organs, specifically the liver, lungs, and kidneys, were harvested for histological analysis. These organs were preserved using 10% neutral buffered formalin at different temperatures and underwent tissue processing. The fixation was conducted with 10% neutral buffered formalin at room temperature (25\u0026deg;C, group 1), at 37\u0026deg;C (group 2), and at 60\u0026deg;C (group 3) for a period of 24 hours, followed by dehydration through various grades of alcohol (50%, 70%, 80%, and 90%) and two changes of absolute alcohol sourced from Sigma-Aldrich, each lasting one hour [2]. Subsequently, the dehydrated tissues were cleared with two changes of toluene (Surgipath Medical Industries, Inc.), also for one hour each, and infiltrated with paraffin wax over three changes, each lasting one hour. The processed tissue sections were embedded in paraffin wax, and the resulting paraffin-embedded blocks were sectioned using a hand rotary microtome (ERM 3100 Hestion Histology Equipment). The sections were mounted onto glass slides (Unifrost microscope slide), then deparaffinized and rehydrated before being stained as outlined below. Stained sections were examined using an Olympus binocular light research microscope and recorded with a DXM1200 digital camera (Nikon).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of Slides\u003c/h2\u003e \u003cp\u003eLiver, lung, and kidney tissue sections that had been processed with paraffin wax were prepared for staining. Hematoxylin and Eosin (H and E) was the chosen staining protocol. All groups of slides underwent staining with Hematoxylin and Eosin. The slides were de-waxed in xylene for approximately five minutes and subsequently subjected to progressive grades of alcohol (100%, 90%, 70%, and 50%). Following a rinse in water, the sections were stained with Hematoxylin for around 15 minutes before rinsing again. A differentiation step using 1% acid alcohol for two seconds was performed, followed by another rinse in water. The sections were blued with tap water for ten minutes and stained with Eosin for 30 minutes. Excess staining was eliminated through dehydration with graded alcohol (50%, 70%, 90%, and 100%). The final stage involved clearing the slides in xylene and mounting them using DPX. The duplicates of the slides were analyzed utilizing an ATR-FTIR spectrometer. This spectrometer facilitated direct analysis of the sections, identifying similarities and discrepancies while assessing the biomolecules within the tissues that were either retained or lost, according to Adeleke et al [34].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Fourier Transform Infrared (ATR-FTIR) Spectroscopy Methodology\u003c/h2\u003e \u003cp\u003eFTIR spectra of all the analyzed samples were obtained using the Agilent Cary 630 spectrometer from Agilent Technologies. The Attenuated Total Reflectance (ATR) technique utilized a diamond crystal for measurements taken across the IR spectrum from 4000 to 600 cm-1, employing 32 scans at a resolution of 16 cm-1. Each measurement was repeated five times. The resultant spectra underwent normalization, smoothing, and baseline corrections via Spectagyrph software [34].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical Analysis\u003c/h2\u003e \u003cp\u003eData analysis of FTIR results was conducted employing IBM SPSS Statistics 25 software, with results presented in table format as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. An inferential statistic, specifically the t-test, was utilized for comparisons between two groups. Additionally, a Receiver Operating Characteristic (ROC) curve was employed to evaluate the performance, sensitivity, and specificity of the methodologies used in comparing temperature-controlled tissue groups. A significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was established for the statistical analysis, with results also conveyed in a tabular format.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Histological Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 1a-c Shows photomicrograph of formalin-fixed Liver tissue in 25\u0026deg;C, 37\u0026deg;C, and 60\u0026deg;C respectively.\u003c/p\u003e\n\u003cp\u003eFigure 2a-c Shows photomicrograph of formalin-fixed Lungs tissue in 25\u0026deg;C, 37\u0026deg;C, and 60\u0026deg;C respectively.\u003c/p\u003e\n\u003cp\u003eFigure 3a-c Shows photomicrograph of formalin-fixed Kidney tissue in 25\u0026deg;C, 37\u0026deg;C, and 60\u0026deg;C respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Peak assignment for alpha helix/Beta sheet and their relative spectral signal strength at different temperatures\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"529\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003ePeak Assignment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 164px;\"\u003e\n \u003cp\u003eSecondary protein structures\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u0026nbsp;References\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 201px;\"\u003e\n \u003cp\u003eSignal strength\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 328px;\"\u003e\n \u003cp\u003eLiver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e25\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e37\u003c/strong\u003e\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e60\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e1654\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 164px;\"\u003e\n \u003cp\u003e\u0026alpha; - helix\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 86px;\"\u003e\n \u003cp\u003e39, 40\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eModerate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrong\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eWeak\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e1625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 164px;\"\u003e\n \u003cp\u003e\u0026beta; -sheet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eModerate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrong\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eWeak\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 328px;\"\u003e\n \u003cp\u003eLung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e1654\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 164px;\"\u003e\n \u003cp\u003e\u0026alpha; - helix\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e39, 40\u003c/p\u003e\u0026nbsp;\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrong\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003eWeak\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eModerate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e1625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 164px;\"\u003e\n \u003cp\u003e\u0026beta; -sheet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrong\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003eWeak\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eModerate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 328px;\"\u003e\n \u003cp\u003eKidney\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e1654\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 164px;\"\u003e\n \u003cp\u003e\u0026alpha; - helix\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 86px;\"\u003e\n \u003cp\u003e39, 40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eWeak\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrong\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eModerate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e1625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 164px;\"\u003e\n \u003cp\u003e\u0026beta; -sheet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eWeak\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrong\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eModerate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Comparing \u0026alpha;-helix /\u0026beta; -sheet values of different organs at paired temperature ranges\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"512\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 512px;\"\u003e\n \u003cp\u003e\u0026alpha; \u0026ndash; helix /\u0026beta; -sheet Ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" style=\"width: 59px;\"\u003e\n \u003cp\u003eOrgans\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 152px;\"\u003e\n \u003cp\u003eMean\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"4\" style=\"width: 120px;\"\u003e\n \u003cp\u003e95%Confidence Interval\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 59px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 152px;\"\u003e\n \u003cp\u003eTemperature range\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 152px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 152px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e25 \u0026nbsp;ͦC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e37 \u0026nbsp;ͦC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003eMean difference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003eLower\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eUpper\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003et-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003eLiver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e1.02\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.79\u0026plusmn;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0.229\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0.232\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e479.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003eLung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e1.03\u0026plusmn;0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e1.14\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e-0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e-0.111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e-0.107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e-130.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003eKidney\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.81\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.86\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e-0.0491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e-0.051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e-0.048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e-84.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 59px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 61px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 59px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e25 \u0026nbsp;ͦC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e60 \u0026nbsp;ͦC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003eLiver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e1.02\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.85\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.169\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0.168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0.171\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e260.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003eLung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e1.03\u0026plusmn;0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.92\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0.108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0.112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e116.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003eKidney\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.81\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.86\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e-0.049\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e-0.051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e-0.048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e-84.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e37 \u0026nbsp;ͦC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e60 \u0026nbsp;ͦC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003eLiver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.79\u0026plusmn;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.85\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e-0.061\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e-0.062\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e-0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e-105.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003eLung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e1.14\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.92\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.219\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0.218\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e416.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003eKidney\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.86\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.86\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e-0.0004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e-0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e0.497\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026alpha; \u0026ndash; helix /\u0026beta; -sheet ratio \u0026nbsp;showed statistical difference to distinguish tissues fixed at different temperature except for kidney at 37\u0026deg;C and 60\u0026deg;C \u0026nbsp;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Performance of \u0026alpha; \u0026ndash; helix /\u0026beta; -sheet ratio as a metric of protein conformations on tissues formalin-fixed between 25\u0026deg;C and 37\u0026deg;C\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"623\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 125px;\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;Tissues\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003eTemp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eArea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eSE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003eLower bound\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003eUpper bound\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eSensitivity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eSpecificity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003eCut-off\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003eLiver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0.500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0.255\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.745\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e71%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e0.00*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003eLung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e#1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e#1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003eKidney\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0.462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e0.138\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0.191\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.733\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e46%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e46%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 59px;\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eExploring the utility of \u0026alpha; \u0026ndash; helix /\u0026beta; -sheet ratio as metric of monitoring the effect of increasing formalin temperature (25\u0026deg;C- 37\u0026deg;C) on the structural integrity of proteins during fixation using AUC values, it was observed that only lung that showed potential as a experimental model to study this temperature impact, with \u0026nbsp; \u0026alpha; \u0026ndash; helix /\u0026beta; -sheet showing excellent model performance (1.00), sensitivity (100%), specificity (100%) at statistical significance level (*) as shown in supplementary \u003cstrong\u003efig\u003c/strong\u003e. \u003cstrong\u003e4\u003c/strong\u003e. #Excellent diagnostic performance. ROC, receiver operating characteristics; AUC, area under the curve; CI, confidence interval; Temp, Temperature; SE, standard error. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e ROC Performance of \u0026alpha; \u0026ndash; helix /\u0026beta; -sheet ratio as a metric of protein conformations on tissues formalin-fixed between 25\u0026deg;C and 60\u0026deg;C\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"623\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 141px;\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;Tissues\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003eTemp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003eArea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eSE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003eLower bound\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003eUpper bound\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003eSensitivity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003eSpecificity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eCut-off\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eLiver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e0.500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.134\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e0.238\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e0.762\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e83%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.01*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eLung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e#1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e#1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eKidney\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e71%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.01*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e0.462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.138\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e0.191\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e0.753\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e46%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eExploring the utility of \u0026alpha; \u0026ndash; helix /\u0026beta; -sheet ratio as metric of monitoring the effect of increasing formalin temperature (25\u0026deg;C- 37\u0026deg;C) on the structural integrity of proteins during fixation using AUC values, it was observed that only lung that showed potential as a experimental model to study this with \u0026nbsp;\u0026alpha; \u0026ndash; helix /\u0026beta; -sheet showing excellent model performance (1.00), sensitivity (100%), specificity (100%) at statistical significance level (*) as shown in supplementary \u003cstrong\u003efig\u003c/strong\u003e. \u003cstrong\u003e5\u003c/strong\u003e. #Excellent performance. AUC, area under the curve; ROC, receiver operating characteristics; CI, confidence interval; Temp, Temperature; SE, standard error. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e ROC Performance of \u0026alpha; \u0026ndash; helix /\u0026beta; -sheet ratio as a metric of protein conformations on tissues formalin-fixed between 37\u0026deg;C and 60\u0026deg;C\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"623\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 141px;\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;Tissues\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003eTemp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003eArea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eSE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003eLower bound\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003eUpper bound\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eLiver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e71%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.01*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e85%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.01*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eLung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e#1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e#1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eKidney\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e0.467\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e0.214\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e0.719\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e47%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e0.467\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e0.214\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e0.719\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e47%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eExploring the utility of \u0026alpha; \u0026ndash; helix /\u0026beta; -sheet ratio as metric of monitoring the effect of increasing formalin temperature (37\u0026deg;C- 60\u0026deg;C) on the structural integrity of proteins during fixation using AUC values, it was observed that only lung that showed potential as a experimental model to study this with \u0026nbsp;\u0026alpha; \u0026ndash; helix /\u0026beta; -sheet showing excellent model performance (1.00), sensitivity (100%), specificity (100%) at statistical significance level (*) as shown in supplementary \u003cstrong\u003efig\u003c/strong\u003e. \u003cstrong\u003e6\u003c/strong\u003e. #Excellent performance. ROC, receiver operating characteristics; AUC, area under the curve; CI, confidence interval; Temp, Temperature; SE, standard error.\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThrough the elevation of fixation temperature using standard formaldehyde fixative, this current research sought to examine how different temperature ranges affect both fixation effectiveness and the preservation of tissue architecture for histological examinations. The study aimed to evaluate how modifications in the temperature of the formaldehyde fixative influence specific histological specimens while exploring the associated morphological features, particularly regarding proteins that may either be preserved or transformed in response to heated fixatives.\u003c/p\u003e\n\u003cp\u003eTissue samples from the liver, lungs, and kidneys of Wistar rats were utilized for this investigation. After the organs were extracted, they were subjected to fixation in formaldehyde at temperatures of 25\u0026deg;C, 37\u0026deg;C, and 60\u0026deg;C, respectively. In the case of the liver tissue, hepatocytes were distinctly visible with minimal distortion at the 25\u0026deg;C setting. However, at 37\u0026deg;C, the fixation was inadequate, leading to uneven staining, particularly observed in the cellular centers [33]. Tissue architecture was notably preserved under the 25\u0026deg;C conditions, while at 60\u0026deg;C, the central vein was clearly outlined, though the cytoplasm of the liver cells was singularly observed with nuclei appearing shriveled (refer to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Observations for lung tissue indicated that air spaces underwent greater distortion under the 60\u0026deg;C condition compared to 37\u0026deg;C, suggesting that elevated heat induced denaturation, resulting in marked shrinkage and distortion associated with the lung structure. Nonetheless, the tissue matrix exhibited superior quality at 25\u0026deg;C compared to the higher temperatures, while staining qualities were distinctly differentiated at both 25\u0026deg;C and 37\u0026deg;C [35]. At 60\u0026deg;C, the central zones of the tissue were inadequately fixed, with significant loss of cellular and nuclear definition (as depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) [36]. In the kidney specimens, those fixed at 25\u0026deg;C displayed clear boundaries following staining, contrasting with the 37\u0026deg;C samples, which exhibited greater stain uptake but also maintained general architectural integrity. Conversely, at 60\u0026deg;C, noticeable tears and distortions were present, creating irregular spaces within the tissue architecture\u0026mdash;an observation absents in samples fixed at 25\u0026deg;C and 37\u0026deg;C (as illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) [35]. According to Bamisi and Alese, elevating the fixative temperature enhances the diffusion rate into the tissue, thus accelerating the reactive interactions between tissue and fixative. Nonetheless, extensive investigations into formaldehyde fixation mechanisms have established the concept of \u0026ldquo;rapid penetration, slow fixation\u0026rdquo; [36]. This observation implies that in the present study, the application of heat initiated the fixation process without providing sufficient penetration of the fixative. High temperatures were found to induce tissue fixation through thermal coagulation rather than enhancing the cross-linking process of formaldehyde [37]. The action mechanism of formaldehyde indicates that it penetrates swiftly (as methylene glycol) while the actual fixation is a slower process (as carbonyl formaldehyde) [9]. Therefore, the findings of this investigation propose that while heat facilitated a quicker reaction between tissue and fixative, it did not enhance the penetration rate. Overall, results indicate that while thermal conditions can accelerate fixation rates, they also correspondingly accelerate autolytic processes. It seems fixation proper only commenced once adequate penetration was achieved [3].\u003c/p\u003e\n\u003cp\u003eAdditionally, the investigation into the effects of varying formalin temperatures on tissue specimens revealed a significant influence on the secondary structures of proteins, as evidenced by Fourier Transform Infrared Spectroscopy (FTIR) analyses. The predominant secondary structural motifs identified in proteins, namely alpha helices and beta sheets, are ubiquitous across various tissues in the body [38\u0026ndash;39]. Research suggests that formalin fixation induces folding and denaturation, particularly affecting globular proteins characterized by alpha-helical structures due to the prevalent collagen found in soft tissues, while some proteins remain largely unaffected [10, 13, 20\u0026ndash;21]. Some researchers argue that heat modifies the alpha-helix structures present in normal tissues into beta-sheet conformations, which are often indicative of tissue disorganization [10, 40\u0026ndash;41]. However, the majority of studies examining this phenomenon have focused on the independent impacts of formalin and heat on protein models, with only a few studies evaluating their synergistic effects on proteins like RNAase A, bovine serum albumin, hemoglobin, and ovalbumin.\u003c/p\u003e\n\u003cp\u003eCritical organs such as the liver, lungs, and kidneys are known for their extensive collagen fiber content. The liver, as the largest organ with its five lobes, plays vital roles in metabolism, transport, and detoxification. The kidneys are essential for homeostasis, facilitating ion transport and filtration functions, while the lungs enable gas exchange necessary for respiration [43]. These organs predominantly comprise globular proteins, predominantly alpha-helices interlaced with beta-sheets, which are essential for their various physiological functions.\u003c/p\u003e\n\u003cp\u003eIn supplementary Figs.\u0026nbsp;1 and 2, the observed shift in protein bands corresponding to Amide I (1625 cm-1) and Amide II (1520 cm-1) towards beta-sheet conformations provides evidence of temperature\u0026apos;s impact on protein structure. Notably, more pronounced peaks were found at 25\u0026deg;C and 37\u0026deg;C in comparison to a decrease in intensity at 60\u0026deg;C. Specifically, liver and lung samples fixed at 60\u0026deg;C displayed weaker absorbance compared to those treated at 25\u0026deg;C and 37\u0026deg;C, indicating potential unfolding and dissociation associated with the loss of intermolecular hydrogen bonds within the beta-sheet structures. Interestingly, the lung tissue fixed at 25\u0026deg;C showed more intense protein peaks than tissue fixed at 37\u0026deg;C, aligning with previous studies that indicated a greater binding rate of formalin at 37\u0026deg;C versus 25\u0026deg;C. This leads to the hypothesis of ongoing protein conformational changes occurring at lower temperatures rather than at elevated temperatures like 60\u0026deg;C [37]. The weak intensity of beta-sheets observed at 60\u0026deg;C suggests potential conformational alterations in proteins, with some scholars attributing this to heat coagulation rather than formalin fixation [37]. While the overall implications of this hypothesis have yet to be thoroughly investigated, the present findings revealed a marked reduction in peak intensity for lung tissues fixed at 60\u0026deg;C compared to those fixed at 37\u0026deg;C, indicating possible protein structural distortion. This phenomenon may be attributed to the activation of cellular enzymes at elevated temperatures, which can lead to cellular degradation in enzyme-rich tissues such as the liver [15\u0026ndash;16]. These observations are consistent with similar research advocating for formalin fixation at higher temperatures but over shorter durations [37].\u003c/p\u003e\n\u003cp\u003eConversely, the kidneys fixed in formalin at 60\u0026deg;C exhibited stronger protein peaks at Amide I (1630 cm-1) and Amide II (1530 cm-1), corresponding to beta-sheet structures, followed by those fixed at 25\u0026deg;C, which also outperformed 37\u0026deg;C fixed kidney tissues in peak intensity. Evidence from kidney samples indicates significant protein conformational changes occurring at 60\u0026deg;C, as reflected by the pronounced beta-sheet spectra, suggesting enhanced stability of internal molecular bonds necessary for subsequent tissue processing compared to 25\u0026deg;C and 37\u0026deg;C fixed samples (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea-c).\u003c/p\u003e\n\u003cp\u003eThe observed effects of temperature on protein structure appear inconsistent; while higher temperatures promote tissue stability in the kidneys, detrimental outcomes of heat on the fixation processes, particularly noted in liver and lung tissues, have also been documented [15]. This study suggests that fixation at elevated temperatures may adversely affect liver and lung tissues more than those at room temperature, potentially due to activation of cellular enzymes at elevated temperatures leading to improper fixation prior to spectral analysis. The spectral data for the liver indicates that both Amide I and Amide II peaks corresponding to beta sheets reflect a tissue-formalin interaction, albeit subtly as shown in supplementary Fig.\u0026nbsp;1\u0026ndash;3. This finding contrasts with reports asserting that formalin penetrates liver tissue slowly [15]. Consequently, fixation at higher temperatures likely compromises native protein retention and is unnecessary for effectively preserving cell-to-cell relationships within the tissue matrix [15].\u003c/p\u003e\n\u003cp\u003eAgain, in this study, we examined the relative abundance of native alpha-helices compared to beta-sheets in proteins at 25\u0026deg;C as the temperature increases across liver, lung, and kidney tissues. The ambient temperature of 25\u0026deg;C is frequently cited as optimal for most routine histological analyses [22, 44]. Our results indicated that at this temperature, alpha-helices were predominantly present in liver and lung tissues, while in the kidney, beta-sheets were more prevalent, as illustrated in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and Figs. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. The underlying factors contributing to the elevated beta-sheet presence in kidney tissues remain somewhat ambiguous, but it is likely influenced by formalin-induced conformations. This suggests that the conversion rates from alpha-helix to beta-sheet in lung and liver tissues are relatively stable at room temperature when compared to kidney tissues. Nonetheless, both liver and kidney tissues exhibited a decrease in alpha-helix content relative to beta-sheets at higher temperatures (37\u0026deg;C and 60\u0026deg;C), indicating the significant role of elevated temperatures in progressive entropy leading to protein denaturation [18, 23\u0026ndash;24, 41, 45]. This observation aligns with multiple studies that have pointed to heat as a factor in the transition from alpha-helices to beta-sheets. The increase in beta-sheets has been linked to both direct and indirect transformations originating from the unfolding of alpha-helices and random coils to anti-parallel beta-sheets [27, 40, 46]. Conversely, lung tissue maintained a relatively stable alpha-helix content at 37\u0026deg;C compared to the beta-sheet content. This phenomenon may suggest that 37\u0026deg;C presents a limiting condition for the conversion of alpha-helices to beta-sheets while simultaneously promoting the conversion of unordered random coil structures to alpha-helices [27]. The overarching conclusion of this research indicates that with the progressive increase in formalin temperature from 37\u0026deg;C to 60\u0026deg;C, the beta-sheet content becomes more pronounced compared to alpha-helices, highlighting the notion of heat-induced changes in secondary structure motifs observed across all organs [45]. The basis for these alterations is likely due to the synergistic and interactive influences of formalin and heat on the histological tissues, leading to protein unfolding via the destabilization of hydrophilic bonds within the tissue matrix [22, 42, 44, 47].\u003c/p\u003e\n\u003cp\u003eImportantly, the increase in formalin temperature from 25\u0026deg;C to 37\u0026deg;C and 60\u0026deg;C had minimal effect on the protein substructures of compact tissues like liver and kidney, as measured by the alpha-helix/beta-sheet ratio, which assessed the sensitivity of these tissues to temperature fluctuations. The relative statistical analyses indicated a greater presence of alpha-helices at 25\u0026deg;C in comparison to 60\u0026deg;C, where beta-sheets predominated (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). This finding implies conformational changes wherein normal tissues, under specific favorable conditions, transition to a more destabilized state in pathological scenarios induced naturally or artificially [38, 48]. The cut-off points also suggest higher values at 25\u0026deg;C compared with values at 37\u0026deg;C and 60\u0026deg;C is key to identifying realistic effects especially on liver and lungs. However, only lung tissues exhibited significant changes, demonstrating a measurable impact of fixation temperature on protein secondary structures, as the calculated ratios revealed not only statistical significance but also superior performance across tissues fixed at varying temperatures (Tables \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). This aligns with previous findings where the same ratio was effectively used to investigate protein conformational changes in breast cancer with remarkable accuracy, achieving sensitivity and specificity above 90% [38, 49]. The pronounced effect of formalin temperature on lung tissues, as indicated by this innovative metric, arises from its more loosely arranged parenchyma containing air spaces. The connective tissues in the lungs are thinner and more elastic compared to the denser, fibrous structures of the kidney and liver, which are optimized for metabolic and filtration functions needing a compact surface area [43]. Consequently, the diffusion and penetration of formalin within the lung cavities is enhanced by elevated temperature, leading to corresponding alterations in protein structures, a phenomenon that was less pronounced in the other tissues evaluated for temperature-related protein changes.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e4.1 Potential Consequences of Heat-Induced Conversion from \u0026alpha;-Helix to \u0026beta;-Sheet for Epitope Retrieval\u003c/h2\u003e\n \u003cp\u003eEpitope retrieval is a critical procedure for formalin-fixed and cross-linked tissues, employing heat and enzymatic techniques [50]. This process is based on the principle that the cross-linking of surface proteins can be reversed under specific conditions, including temperature, pH, concentration, and buffer composition [51]. Notably, while formalin alters certain surface proteins, it can also leave some regions untouched, resulting in partial fixation or peripheral fixation [10, 21, 24, 50]. The varying degrees of disruption to tertiary protein structures have been associated with factors like solvent polarity, pH, concentration, duration, and temperature [13, 21, 27]. Traditionally, the optimal temperature for most histological techniques ranges from 20\u0026ndash;25\u0026deg;C [18]. Within this temperature range, the preservation of both secondary and tertiary protein conformations during formalin fixation is typically reversible through appropriate interventions. Numerous studies have investigated the effects of 4\u0026deg;C and 25\u0026deg;C in histological, histochemical, and immunohistochemical contexts [10, 19, 23]. However, temperatures above 40\u0026deg;C can compromise tissue integrity despite enhancing penetration. For example, formalin at 40\u0026deg;C tends to fix tissues peripherally, leaving the central regions largely unaffected, and this phenomenon has been correlated with diminished immunoreactivity [23]. Consequently, conditions that impair or weaken immunoreactive sites on tissue diminish the likelihood of successful epitope recognition by antibodies [12, 24]. Nonetheless, the simultaneous effects of heat and formalin on secondary protein structures and their implications for epitope retrieval and antibody recognition in immunohistochemistry remain incompletely understood. Thus, this section endeavors to conceptually elucidate the mechanisms behind these temperature-induced protein alterations and their unintended consequences on antigen retrieval and antibody binding.\u003c/p\u003e\n \u003cp\u003eFormalin primarily interacts with lysine and cysteine residues, leading to the formation of methylol groups, which subsequently react with other nucleophiles such as tyrosine, glutamine, histidine, arginine, and tryptophan [20, 50]. These methylol groups, also referred to as methylene bridges, are integral to the structural integrity of tissues [22]. A range of studies has explored the impact of heated formalin on non-native protein models [20, 24, 25\u0026ndash;26, 44]. Typically, N-terminal amino acid residues are less reactive with formalin, while side chains of amino acids like cysteine, histidine, lysine, and arginine demonstrate higher reactivity in native protein contexts [10, 50]. Lysine and cysteine, being the most accessible, are likely to undergo significant conformational changes, forming intermolecular crosslinks in response to formaldehyde [10, 26, 51\u0026ndash;52]. The diverse reactivity of these exposed groups on protein surfaces is crucial for epitope recovery and antibody recognition [10, 13, 52]. The retrieval of thermally stabilized crosslinks, or antigen retrieval, can be achieved through heating at 65\u0026deg;C and enzymatic treatments [10, 26]. The ability to revert cross-linked proteins to their native forms under normal conditions indicates no irreversible destruction has occurred. Adhering to the prevailing consensus that increased temperatures induce conformational shifts from alpha-helix to beta-sheet structures in proteins\u0026mdash;due to destabilization of critical hydrogen bonds\u0026mdash;it can be posited that alpha-helices, commonly found in amino acids such as lysine within native proteins, retain their structural and cross-linking associations even after formalin treatment, allowing for their reversion when favorable conditions are met [27, 40, 44, 46]. Nevertheless, observations from this study suggest that elevated temperatures might prompt irreversible conversions of alpha-helical amino acids to beta-sheet structures, particularly in long-chain poly-L-lysine native tissues, regardless of optimal conditions and retrieval methods [27]. This heat-induced transformation is attributed to the gauche isomerization of CH2 groups in the hydrocarbon side chains of lysine amino acids within both reactive and non-reactive tissue domains [27, 41, 50]. Increased temperatures have been shown to accelerate the conversion of alpha-helices to beta-sheets, the latter known for their comparatively weakened exposed intermolecular hydrogen bonds, resulting in enhanced thermal stability but reduced digestibility [40, 42, 44]. Consequently, this alteration renders tissues less susceptible to subsequent interactions, such as antibody adherence. Alpha-helices demonstrate more local mobility within their native pools compared to the relatively rigid beta-strands, which typically require additional energy for unfolding. This phenomenon may lead to a transient-post permanent loss of essential immunoreactive sites necessary for antibody binding, leaving only protein components unaffected by formalin or heat to interact with antibodies, often resulting in weak or absent staining visibility [20, 24, 50]. Additionally, amino acids that are known to exhibit beta-sheet conformations, such as isoleucine, tyrosine, tryptophan, and valine, are secondarily influenced by formalin, lending support to the hypothesis that the transformation from alpha-helix to a fundamentally different conformation, such as beta-sheet, alters protein surfaces, revealing amino acids that are not characteristic of the native protein but emerge as byproducts of heated formalin interactions affecting protein structural integrity [53].\u003c/p\u003e\n\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eWhile various methods, including thermogram and differential scanning calorimetry and circular dichroism, have been employed, they are often considered less sensitive to minor transitions in protein denaturation at the secondary structure level [25]. However, Fourier-transform infrared (FTIR) spectroscopy not only captured the qualitative and quantitative changes induced by increased formalin temperature relative to the standard 25\u0026deg;C, but the derivative values from the alpha-helix/beta-sheet ratio subjected to statistical analysis revealed that it could be utilized to detect changes in protein conformation as temperature rises, which may affect downstream applications like immunohistochemistry. This research indicates that Fourier transform infrared spectroscopy (FTIR) can provide a comprehensive method for examining the impact of thermal exposure on proteins, applicable at various stages of laboratory processes, particularly during the analytical phase of tissue preparation, which encompasses fundamental procedures like tissue fixation. The ratio of α-helix to β-sheet structures has demonstrated considerable sensitivity in identifying changes at the protein level, especially in loosely organized tissues such as lung tissue, with a commendable degree of precision. This advancement signals the potential incorporation into quality management systems within the histopathology domain, promising significant improvements in the reliability and validity of real-time histopathological diagnoses.\u003c/p\u003e"},{"header":"6 Future Directions","content":"\u003cp\u003eWhile it is believed that rat anatomical characteristics approximates that of human, future studies on real human samples would be commendable. It is also advisable for forthcoming research to utilize the α-helix to β-sheet ratio as a parameter to assess the thermal effects utilizing a larger sample size. Additionally, examining temperature elevations beyond those studied here in solid organs may prove beneficial in evaluating the efficacy of this innovative marker in various contexts. While the lung has been established as an excellent model for investigating the consequences of high-temperature formalin fixation, other tissue types should be considered in future studies to broaden the scope of this research. The use of image analysis and artificial intelligence might also bring new insights into patterns of formalin-temperature-induced changes on organs opening up a roadmap to empirical quality management system in histopathological laboratories.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThe authors did not receive any research grant or fund from any place or organization for the completion of this present study\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthors contributions AST: Conceptualization, Supervision, Data analysis and interpretation, Manuscript writing and editing; AEA; Data collection, first draft writing; AOG: Data collection, Manuscript review; IC: Manuscript revision\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll necessary data supporting our findings for this present study are enclosed within the manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHowat WJ, and Wilson BA. 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Toxicol\u003c/em\u003e\u003cstrong\u003e25\u003c/strong\u003e(2): 95-100.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Formaldehyde, Temperature, Fixation, Motifs","lastPublishedDoi":"10.21203/rs.3.rs-8773932/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8773932/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFormaldehyde is a commonly used fixative in histological studies, with fixation outcomes significantly influenced by physical factors such as temperature. This study investigates the impact of varying fixation temperatures on tissue morphology and protein structural integrity using formaldehyde as the fixative. Ten (10) Wistar rats were sacrificed via cervical dislocation, and their liver, lungs, and kidneys were extracted. These organs were fixed in 10% Neutral Buffered Formalin at 25\u0026deg;C, 37\u0026deg;C, and 60\u0026deg;C for 24 hours. Standard tissue processing was followed, with 4 \u0026micro;m sections prepared for histological analysis using hematoxylin and eosin staining to assess tissue morphology. Additionally, 20 \u0026micro;m sections were analyzed using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy to examine protein spectral characteristics, employing the Agilent Cary 630 spectrometer in the 4000\u0026ndash;600 cm⁻\u0026sup1; IR range.\u003c/p\u003e \u003cp\u003eResults revealed that tissues fixed at 25\u0026deg;C and 37\u0026deg;C showed superior preservation, while those at 60\u0026deg;C exhibited significant distortion for most of the tissues. Findings also showed that there was an elevation in beta-sheets as formalin temperature increases to 60\u0026deg;C compared with alpha-helixes on liver, lungs, and kidneys. However, performance test using receiver\u0026rsquo;s operating characteristics (ROC) curve showed potential of α \u0026ndash; helix /β -sheet ratio to monitor effect of formalin-temperature increase on tissues and lung tissues appeared to be a perfect model for this study showing 100% accuracy, 100% sensitivity and 100% specificity to tell apart changes occuring to protein secondary structures between 25\u0026deg;C controlled formalin fixation and those at higher fixed at 37\u0026deg;C. These findings underscore the importance of optimizing fixation temperature to balance fixation speed and tissue integrity, drawing implications for antigen recovery in immunohistochemical studies.\u003c/p\u003e","manuscriptTitle":"A Novel α – helix /β -sheet ratio as a Potential Metric For Studying Thermal Effects of Increased Formaldehyde Temperatures on Native Tissues","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 10:07:33","doi":"10.21203/rs.3.rs-8773932/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"94346d41-302b-4702-a639-1e83b663872a","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-28T14:24:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 10:07:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8773932","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8773932","identity":"rs-8773932","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}