Standardizing dentate gyrus isolation for molecular adult hippocampal neurogenesis studies: a comparative and tissue softening-enabled workflow

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To our knowledge, the two widely used DG microdissection approaches, medial (intact-block) and coronal (slice-guided) have not been directly compared under matched conditions, and the value of a simple tissue-softening step for operational standardization has not been quantified. To provide a comparative, quantitative validation of the medial and coronal DG microdissection approaches and to establish a tissue softening enabled workflow that laboratories can adopt for training and standardization. Adult rat hemispheres were assigned to seven groups (n = 3 hemispheres/group): fresh-medial, fresh-coronal, fixed-medial, fixed-coronal, softened-medial, softened-coronal, and intact control (fixed). A 15-day slow running-water rinse softened archival tissue. Outcomes were (i) operational performance (time to isolate DG) and (ii) anatomical specificity (residual CA1-CA3 area on H&E after the DG removal). In fresh tissue, the medial approach isolated DG in 51.7 ± 6.5 vs 125.3 ± 8.5 s for the coronal approach (~ 2.4× difference). In softened fixed tissue, both approaches were slower, but the speed ranking (medial 0.05). Softening improved border visibility and handling, particularly for the coronal approach, providing an operational surrogate for training and standardization. Under matched conditions, medial approach offers faster procurement while coronal maximizes border visibility; both maintain comparable anatomical specificity. The 15-day softening protocol supports 3R-aligned training and standardization. dentate gyrus adult hippocampal neurogenesis microdissection tissue softening Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The DG, a sub-region of the hippocampus (HP), contains the sub-granular zone, the primary site of AHN (Piatti etl al. 2013; Kempermann et al. 2015 ; Lazarov & Hollands 2015). AHN generates new excitatory principal neurons in an activity-dependent manner, supporting cognitive processes and being implicated in numerous neurological and psychiatric disorders (Goçalves et al. 2016; Moreno-Jiménez et al. 2019 ). Since its discovery by Altman & Das ( 1965 ), AHN has become a central topic in contemporary neuroscience. Immunohistochemistry on sectioned brain tissue remains a cornerstone of AHN research (von Bohlen und Halbach 2011; Kempermann et al. 2004 ). However, deeper molecular investigations such as transcriptomic and proteomic analyses require freshly isolated DG to preserve the integrity of nucleic acids and proteins (Hagihara et al. 2009 ). Accordingly, to preserve spatial gradients and cell-type composition for downstream molecular analyses, AHN research relies critically on precise DG procurement, free from contamination by adjacent hippocampal regions such as CA1-CA3 (Ianov et al. 2017 ; Alkadhi 2019 ). Despite its importance, most published molecular studies lack detailed methodological descriptions or visual documentation of DG dissection, limiting reproducibility and transparency. Only a few methodological studies provide detailed guidance on DG removal (Hagihara et al. 2009 ; Gilley et al. 2011 ; Guo et al. 2012 ). Despite this centrality, the two canonical DG microdissection strategies have never been compared head-to-head under matched conditions. Gilley et al. ( 2011 ) and Guo et al. ( 2012 ) described a technique involving serial coronal sectioning and excision of DG in fragments. In contrast, Hagihara et al. ( 2009 ) introduced an approach from the medial surface of the hemisphere that permits the removal of the DG as an intact tissue. In this study, we refer to these as the coronal and medial approaches, respectively. In this context, we directly compared the two canonical DG microdissection approaches and established a practical, adoptable workflow that can be implemented using paraformaldehyde (PFA)-fixed archival brains commonly available in neuroscience laboratories. Because long-term fixed tissue is often considered suboptimal for microdissection, owing to fragility and loss of natural color/contrast needed for neuroanatomical border visibility, we employed a simple 15-day slow-running-water rinse to soften the tissue and restore landmark visibility, thereby enabling precise DG isolation from existing repositories. We then performed a side-by-side validation of the medial and coronal strategies across fresh, fixed, and softened-fixed rat hemispheres, quantifying (i) time to isolate the DG and (ii) residual CA1-CA3 area on histology after DG removal as an objective measure of anatomical specificity. By grounding the study in a direct comparison of two widely referenced methods (Hagihara et al. 2009 ; Gilley et al. 2011 ) and incorporating a feasible softening step for archival tissue, we provide a reproducible procurement framework that enhances molecular precision and cross-study comparability in AHN research, while also reducing reliance on new animals as a secondary benefit. Materials and Methods Ethics All procedures complied with institutional and national guidelines and were approved by the Mersin University Ethics Committee (2016/09). Animals We used 21 adult female Sprague Dawley rats (6–7 months; ~190–210 g). For fixed cohorts, rats were perfused with saline followed by ice-cold 4% PFA in 0.1 M PBS. Brains were immersion-fixed for 48 h in 4% PFA, then stored 4 years at 4°C in 0.1–0.5% PFA in PBS. Fresh hemispheres were obtained under deep ketamine/xylazine anesthesia. Experimental Design We designed seven groups (n = 3 hemispheres/group) were defined: 1) Control (no DG removal) a positive control group in the histological assessment of DG dissection success; 2) Fixed-Medial (FXM), PFA fixed hemispheres, dissected with the medial approach; 3) Fixed-Coronal (FXC), PFA fixed hemispheres, dissected with the coronal approach; 4) Softened-Medial (SM), PFA fixed hemispheres that subjected to softening procedure, dissected with the medial approach; 5) Softened-Coronal (SC), PFA fixed hemispheres that subjected to softening procedure, dissected with the coronal approach ; 6) Fresh-Medial (FM), freshly held hemispheres, dissected with the medial approach; 7) Fresh-Coronal (FC), freshly held hemispheres, dissected with the coronal approach (Fig. 1 ). Brain Hemisphere Softening Procedure To improve the texture and handling of PFA-fixed rat brain hemispheres, we leached residual fixative using a continuous, slow-running tap water rinse, verifying the flow several times daily. In pilot trials, using restoration of hippocampal color contrast and tactile pliability as endpoints, 15 days yielded the best balance; this duration was therefore adopted for the softening protocol. DG Dissection via Medial Approach We adapted the medial side dissection protocol from Hagihara et al. ( 2009 ) (Supplementary Videos 1–3, Fig. 3 ). DG Dissection via Coronal Approach We adapted the coronal slice dissection protocol from Gilley et al. ( 2011 ) (Supplementary Videos 4–6; Fig. 4 ). Prior to dissection, each brain hemisphere was placed on a stable platform positioned over an ice bucket, and 600 µm-thick coronal slices were rapidly sectioned (Fig. 4 A). After the sectioning We utilized the following steps that are also illustrated in a schematic (Fig. 4 ). Vibratome Sectioning We performed vibratome sectioning either prior to applying the medial approach or following the coronal dissection, depending on the study group (as summarized in the experimental design; see Fig. 1 ). The procedure was adapted from previously published protocols (Öztürk & Koç 2022 ; Öztürk et al. 2017 ). We embedded brain hemispheres in 1% (w/v) agarose and sectioned into 600 µm thick coronal slices using a vibratome (Leica Microsystems, Buffalo Grove, IL) (see Supplementary Video 7). Outcome measures Operational performance: time (seconds) to complete DG isolation from one hemisphere. Anatomical specificity: residual CA1-CA3 surface area on H&E-stained coronal sections after DG removal, quantified using the freehand selection tool in ImageJ, National Institutes of Health, Bethesda, MD; https://imagej.nih.gov/ij ) Hematoxylin and Eosin Staining To assess the area sizes of CA regions (1–3) in the DG-excised brain hemispheres, we performed H&E staining with 600 µm thick coronal slices along with the experimental groups. Imaging and Video Capturing To demonstrate the whole, HE stained coronal slices in single piece, we montaged several adjacent images captured by (Olympus, SZ51) light microscope, equipped with an Olympus LC30 digital camera (Olympus Optical Company Ltd, Tokyo, Japan). We created and montaged the videos by using Microsoft Clipchamp application ( https://clipchamp.com/en/video-editor-free online video editor, video compressor, video converter). The original speed of the videos is accelerated by two times. Statistical Analysis We performed statistical analyses with the free trial version of IBM SPSS Statistics 26 software. Initially, we tested the normality and equality of variances of each data set by using Shapiro-Wilk and Levene’s tests, respectively. As we detected that the normality assumption and Levene’s test were not violated, we used independent Samples T test or one-way ANOVA for each of the normally distributed dependent variable. We presented reported values as means ± SD, t(df) and p. Results Visibility and handling in archival tissues When we attempted to practice the coronal (Gilley et al. 2011 ) and medial (Hagihara et al. 2009 ) DG dissection approaches on 4% PFA fixed brain hemispheres with 4 years of archival age, we found that the tissues were thoroughly stiff and fragile which prevented smooth operation of dissection (Supplementary Video 2 and Video 5). We realized that due to the major color alternation on the tissues (Fig. 5 A-C), demarcating the borders of HP from the surrounding structures and sub-HP regions were extremely difficult on the medial surface (Fig. 5 D and 5 E) and on the coronal slices (Fig. 6 B), relative to the fresh brains. Softening restores visibility and handling in archival tissue The 15-day softening restored color contrast and tissue pliability, improving visibility of the hippocampal borders on both medial surfaces and coronal slices. Operational comfort increased, particularly for the coronal approach. At the 15th day of the procedure, we detected significant color change in the HP as compared to the fixed hemispheres (Fig. 5 B and 5 C). On the medial surface of the softened hemispheres, we saw that the borders of HP and surrounding structures became conveniently visible as compared to the fixed brain hemispheres (Fig. 5 E and 5 F). We further observed strong improved visibility of the neuroanatomical borders in the fixed vs softened brains in the coronal slices (Fig. 6 B and 6 C). After the softening procedure, we detected markedly improved operational comfort on coronal approach dissection (Supplementary Video 6), whereas the smoothness of the tissue was still not at the desired level with the medial approach (Supplementary Video 3). We obtained several measurements in the current study. Initially, we tested the normality and equality of variances of each data set by using Shapiro-Wilk and Levene’s tests, respectively. As we detected that the normality assumption and Levene’s test were not violated, we used independent Samples T test or one-way ANOVA for each of the normally distributed dependent variables. Operational performance: medial is faster than coronal In the fresh and fixed softened brains, we recorded the total dissection duration to remove the entire DG from a single hemisphere both with medial and coronal approaches (for descriptive statistics, see Table 1 ). On fresh hemispheres, we found that the medial dissection was significantly faster than the coronal (FM vs FC, 51.67 ± 6.51 s vs 125.33 ± 8.50 s; t(4) = − 11.80, p = 0.00029). The same pattern held for softened-fixed hemispheres (SM vs SC, 301.66 ± 12.34 s vs 727.33 ± 16.62; t(4) = − 35.61, p = 3.71×10⁻⁶) (for independent T test statistics, see Table 2 ). As expected, both approaches were slower in softened-fixed than fresh tissue, both approaches were slower in softened-fixed than fresh tissue (FM vs SM: t(4) = − 31.01, p = 6.43×10⁻⁶; FC vs SC: t(4) = − 55.68, p = 6.22×10⁻⁷). Table 1 Descriptive statistics for DG removal duration (s) Approach FM FC SM SC Total DG removal duration (s) 51.70 (6.51) 125.33 (8.62) 301.66 (12.34) 727.33 (16.62) Total DG dissection durations in seconds among the groups. DG dentate gyrus, FC fresh coronal, FM fresh medial, SM soften medial, SC softened coronal. Table 2 Independent t-test for DG removal duration Variable T(df) p 95% CI FM vs FC -11.80 (4) 0.00029 [-90.95-56.30] SM vs SC -35.61 (4) 3.71x10 − 6 [-458.85-392.47] FM vs SM -31.01 (4) 6.43x10 − 6 [-272.34-227.59] FC vs SC -55.68 (4) 6.22x10 − 7 [-632.01-571.98] Bold font indicates a significant p-value of < .05. CI confidence interval, DF degrees of freedom, FC fresh coronal, FM fresh medial, SM soften medial, SC softened coronal. Anatomical specificity: comparable across strategies and tissue states Lastly, to estimate the success of DG dissection, we measured the area sizes of the CA1-3 regions on the H&E stained coronal slices (600 µm thick) taken after the DG removal procedures. We placed the measurements in the following groups; Control (no DG removal, intact brain hemispheres as regional control) (Fig. 7 A); FM (fresh medial approach) (freshly obtained hemispheres, dissected with medial approach) (Fig. 7 B), FC (fresh coronal approach) (freshly obtained hemispheres, dissected with coronal approach) (Fig. 7 C), SM (softened medial approach) (paraformaldehyde fixed hemispheres that subjected to softening procedure, dissected with medial approach) (Fig. 7 D), SC (softened coronal approach) (paraformaldehyde fixed hemispheres that subjected to softening procedure, dissected with coronal approach) (Fig. 7 E). We found that residual CA1-CA3 areas did not differ significantly across groups (one-way ANOVA, p > 0.05; see Tables 3 – 4 ; Figs. 7 – 8 ), indicating comparable anatomical specificity for medial and coronal strategies whether performed on fresh or softened-fixed tissue. Table 3 Descriptive statistics of CA1-3 area sizes Mean (sd) Control FM FC SM SC CA1 0.27 (0.023) 0.23 (0.02) 0.25 (0.049) 0.16 (0.02) 0.29 (0.03) CA2 0.032 (0.002) 0.023 (0.002) 0.027 (0.008) 0.025 (0.01) 0.036 (0.002) CA3 0.10 (0.014) 0.076 (0.008) 0.11 (0.026) 0.091 (0.037) 0.11 (0.017) CA cornu ammonis, FC fresh coronal, FM fresh medial, SD standard deviation, SM soften medial, SC softened coronal. Table 4 One-way ANOVAs Variable F (df,error) p CA1 2.36 (4,10) 0.123 CA2 0.72 (4,10) 0.597 CA3 0.642 (4,10) 0.644 F Indicates the statistic for ANOVA is the ratio of the mean square for the between groups divided by the mean square within groups. CA cornu ammonis, DF degrees of freedom. Discussion This study provides a head to head validation of two canonical DG microdissection strategies, demonstrating that the medial approach is faster while both medial and coronal approaches achieve comparable anatomical specificity as quantified by residual CA1-CA3 area. By isolating the dissection strategy as the key pre-analytical variable, our results address a long-standing gap that directly impacts the reproducibility and comparability of AHN experiments that depend on precisely procured DG tissue. Medial vs coronal: visibility vs speed In fresh brains, natural color contrast and tissue pliability made both strategies smooth to execute; nonetheless, the medial approach was consistently faster (~ 52 ± 6.5 s) than the coronal approach (~ 125 ± 8.5 s), with high operator comfort for both. By contrast, PFA-fixed tissue was stiff, fragile, and low-contrast, obscuring DG-CA borders and reducing comfort regardless of strategy, an observation consistent with prior emphasis on maintaining clear DG-CA boundaries (Hagihara et al. 2009 ). To mitigate this, a 15-day slow-running-water rinse improved pliability and border visibility. After softening, coronal dissections benefitted the most, with handling and visibility approaching the fresh tissue experience; the medial approach remained workable but was slightly less comfortable than in fresh tissue due to its reliance on precise medial surface landmarks during diencephalon removal. As expected, both strategies took longer in softened-fixed than in fresh tissue, but the relative ranking in speed was preserved (medial < coronal), while anatomical specificity remained comparable across approaches. Taken together, we recommend choosing medial when speed and intact-block removal are priorities, and coronal when maximal border visibility and stepwise control are preferred, especially on softened archival tissue. 3R-aligned training and workflow standardization The 15-day softening protocol provides an excellent operational surrogate for DG microdissection, readily adoptable by many neuroscience labs to standardize technique and workflow for DG and other hard to dissect neuroanatomical regions. This enables training and protocol optimization without excessive new animal use. In line with the 3Rs framework (Replacement, Reduction, Refinement) as articulated by Russell and Burch ( 1959 ) (Russell and Burch 1959 ), this surrogate replaces portions of training that would otherwise require newly sacrificed animals, reduces overall numbers for skill acquisition/pilot work, and refines procedures via improved border visibility and handling. Implications for AHN workflows For AHN studies, precise DG procurement is critical because the DG shows robust dorsal-ventral molecular and epigenetic differentiation (Christensen et al. 2010 ; Zhang et al. 2018 ; Lothmann et al. 2021 ), along with cell and circuit level differences including divergent mossy cell projections and synaptic modulation along the axis (Houseret et al. 2021; Trompoukis & Papatheodoropoulos 2020 ). Variation in dissection boundaries or procedural time can therefore plausibly bias downstream molecular readouts and histological quantification. Our head to head comparison indicates that strategy choice affects speed and operational comfort without compromising anatomical specificity. Although we did not assay molecular endpoints, favoring a medial microdissection when minimizing ex vivo time is the priority and a coronal microdissection when maximal boundary visibility or precise dorso-ventral targeting is required is a logical way to reduce pre-analytical variability in AHN pipelines. This recommendation is motivated by robust dorso-ventral differences in the dentate gyrus (Christensen et al. 2010 ; Zhang et al. 2018 ; Lothmann et al. 2021 ; Houseret et al. 2021; Trompoukis & Papatheodoropoulos 2020 ). Regarding the degradation of nucleic acids and proteins, preferring one of these approaches over another when performed on fresh tissues could be a choice. The storage temperature and duration have an impact on the RNA integrity of brain tissue sections which might be the most critical factors affecting gene expression level in RNA sequencing (Jia et al. 2021 ). However, Catts and colleagues detected no significant change on RNA yield, RNA purity and 28S/18S optical density at the 6th postmortem hour of the mouse brain tissue (Catts et al. 2005 ). Therefore, we expect that the approximately one minute longer duration of the coronal approach would not significantly impact RNA quality. We summarize our study experience to guide researchers; detailed notes are provided in Table 5 . Table 5 Operational comparison of DG microdissection strategies across tissue states and recommended use-cases Tissue state Approach Speed (relative) Operator comfort & border visibility Anatomical specificity (residual CA1–CA3) Recommended use-case Notes Fresh Medial Faster (e.g., ~ 52 ± 6.5 s) High comfort; intact-block removal feasible Comparable to coronal (ANOVA n.s.) Minimize ex-vivo time; rapid procurement for RNA/protein-sensitive workflows Requires confident medial-surface landmarking during diencephalon removal Fresh Coronal Slower (e.g., ~ 125 ± 8.5 s) Excellent slice-face visibility; stepwise control Comparable to medial approach (ANOVA n.s.) Maximal border control; dorsal-ventral sampling strategies Slightly longer procedure; maintain strict cold chain Softened-fixed (15-day rinse) Medial Slower than fresh; faster than coronal (rank preserved) Workable, but comfort lower than fresh due to reliance on medial landmarks Comparable to coronal (ANOVA n.s.) Training/standardization without new animals; pilot histology Operational surrogate; not for molecular assays Only for practicing Softened-fixed (15-day rinse) Coronal Slower than fresh Markedly improved visibility; handling approaches fresh Comparable to medial (ANOVA n.s.) Training; optimizing border definitions; complex borders Best visibility on softened tissue; good for stepwise teaching Fixed (unsoftened) Medial/ Coronal Slowest Low visibility; stiff/fragile, difficult borders Not evaluated for advantage Not recommended; soften first Apply 15-day slow-running-water rinse before use Speed entries highlight the relative rank; example means ± SD are shown for fresh tissue from the present dataset. For softened-fixed tissue, both strategies were slower than fresh, but the medial < coronal speed ranking persisted. Summary of operational performance and anatomical fidelity for medial vs coronal DG microdissection across fresh, softened-fixed, and fixed (unsoftened) tissue states. The medial approach is faster; coronal provides superior visibility, especially after softening. Anatomical specificity is comparable between strategies (ANOVA n.s.) across states. DG dentate gyrus, AHN adult hippocampal neurogenesis, n.s. not significant (p > 0.05). Limitations Sample sizes per group were modest (n = 3), and we did not quantify RNA/protein integrity post-procurement from archival tissue here. Future studies should pair this workflow with RNA/DNA/protein quality control metrics (e.g. RIN and DIN) and extend validation to mouse models and single-nucleus preparations for AHN focused omics. Conclusion We provide a validated, adoptable workflow for DG microdissection that (i) compares medial vs coronal strategies under matched conditions, (ii) demonstrates comparable anatomical specificity across strategies and tissue states, (iii) shows that a 15-day softening rescues archival PFA-fixed brains for precise DG procurement, and (iv) enables molecularly precise AHN studies while limiting additional animal use. Declarations Funding This research was carried out at the Neuroanatomy and Experimental Research Laboratory, which was established with the infrastructure grant support of the Research Projects Unit of Mersin University (Grant No. 2018-1-AP5-2895, 2020-1-AP5-4104). Consumables used in this study were partially used from the grants supported by research grant (116S458) supported by The Scientific and Technological Research Institution of Türkiye. Declaration of Interest All authors declare no conflict of interest. All authors read and approved the final manuscript and attest to the integrity of the original data and the analysis reported in this manuscript. Ethics statement The study has been approved by Ethics Committee of Mersin University (2016/09). Availability of data and materials The data (accessible part) presented in this study is available upon reasonable request to the corresponding author. Author Contributions Conceptualization: [Turan Koç, Nail Can Öztürk]; Methodology: [Turan Koç, Nail Can Öztürk]; Formal analysis and investigation: [Turan Koç, Nail Can Öztürk]; Writing - original draft preparation: [Turan Koç, Nail Can Öztürk]; Writing - review and editing: [Turan Koç, Nail Can Öztürk]; Funding acquisition: [Nail Can Öztürk]; Resources: [Nail Can Öztürk]; Supervision : [Nail Can Öztürk] Acknowledgment The authors acknowledge the use of artificial intelligence-assisted language tools (ChatGPT, Open AI) exclusively for the purpose of enhancing grammar, articulation, and scientific tone. These tools were not used for content generation, data analysis, data interpretation, or any other aspect of scientific authorship. All intellectual content was developed solely by the authors, and the final manuscript was critically reviewed and approved by all contributors. All Supplementary Videos include embedded captions and descriptions. The authors also extend their sincere gratitude to Prof. Zeliha Kurtoğlu Olgunus (former Head of the Department of Anatomy) as well as to the members of the Department of Anatomy, Mersin University Faculty of Medicine, for their contributions to the development of the Neuroanatomy and Experimental Research Laboratory (Project No. 2018-1-AP5-2895). References Alkadhi KA (2019) Cellular and Molecular Differences Between Area CA1 and the Dentate Gyrus of the Hippocampus. Mol Neurobiol 56(9):6566–6580. https://doi.org/10.1007/s12035-019-1541-2 Altman J, Das GD (1965) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol 124(3):319–335. https://doi.org/10.1002/cne.901240303 Catts VS, Catts SV, Fernandez HR, Taylor JM, Coulson EJ, Lutze-Mann LH (2005) A microarray study of post-mortem mRNA degradation in mouse brain tissue. Brain research. 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Supplementary Files GraphicalAbstract..jpg SupplementaryVideo1.mp4 SupplementaryVideo2.mp4 SupplementaryVideo3.mp4 SupplementaryVideo4.mp4 SupplementaryVideo5.mp4 SupplementaryVideo6.mp4 SupplementaryVideo7.mp4 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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10:48:29","extension":"html","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100493,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/3fdc167b2ce142c0f500147a.html"},{"id":92587879,"identity":"316823a0-1510-4ef9-a89f-cb6f699118ce","added_by":"auto","created_at":"2025-10-01 11:04:29","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":116247,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design illustrating the condition of brain hemispheres (\u003cem\u003eN\u003c/em\u003e=3/each group), group codes, and subsequent experimental procedures. “Fixed” refers to post-fixation in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 48 hours following trans-cardiac perfusion with the same fixative. “Fresh” indicates immediate brain removal without fixation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFC\u003c/em\u003e fresh coronal, \u003cem\u003eFM\u003c/em\u003efresh medial, \u003cem\u003eFXC\u003c/em\u003e fixed coronal, \u003cem\u003eFXM\u003c/em\u003e fixed medial, \u003cem\u003eSC\u003c/em\u003esoftened coronal, \u003cem\u003eSM \u003c/em\u003esoftened medial.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e+\u003c/em\u003e Applied, - not applied\u003c/p\u003e\n\u003cp\u003e# Fixed brain hemispheres under slowly running tap water for 15 days\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/4572a88a2dbdd74af64dde89.jpeg"},{"id":92586692,"identity":"9d227061-ba74-4caa-97e6-151de8e71bdb","added_by":"auto","created_at":"2025-10-01 10:48:28","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79605,"visible":true,"origin":"","legend":"\u003cp\u003eStepwise procedures for obtaining brain hemispheres are summarized in panels A–E. Incision sites are indicated by scissor icons and labeled as steps 1–3.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/4d7650dc1aca120f7bfb7baa.jpeg"},{"id":92586693,"identity":"ec50949a-2032-4264-85a4-ee848c5be984","added_by":"auto","created_at":"2025-10-01 10:48:28","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":328103,"visible":true,"origin":"","legend":"\u003cp\u003eStepwise procedures for isolating the dentate gyrus (DG) using the medial approach are illustrated schematically in panels A–C. Sequential images captured from the dissection video are shown in panels D–I. The incision points are indicated with scissor icons in panels A and B.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/17daed57fb87a73636da1c19.jpeg"},{"id":92587634,"identity":"fb250b16-a01f-497c-a842-54c5116a6bde","added_by":"auto","created_at":"2025-10-01 10:56:28","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":353091,"visible":true,"origin":"","legend":"\u003cp\u003eStepwise procedures for isolating the dentate gyrus (DG) using the coronal approach are illustrated schematically in panels A and B. Sequential frames from the dissection video are presented in panels C–J. Each operational step is numbered in panel B and described in the figure. The red asterisk points out the isolated dentate gyrus (DG) tissue (J).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDG\u003c/em\u003e dentate gyrus, \u003cem\u003eCA\u003c/em\u003ecornu ammonis, \u003cem\u003eIB\u003c/em\u003e infra blade, \u003cem\u003eSB\u003c/em\u003e supra blade.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/38bab270d563a3d62a1dadd5.jpeg"},{"id":92586691,"identity":"584f70ab-fea3-4480-8f55-d43b5ebade45","added_by":"auto","created_at":"2025-10-01 10:48:28","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":156268,"visible":true,"origin":"","legend":"\u003cp\u003eLateral and medial surfaces of the brain hemispheres from the fresh, fixed, and softened groups are shown in panels A–C and D–F, respectively. Panels G–I display the medial surfaces of the hemispheres during dissection using the medial approach.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBS\u003c/em\u003e brainstem, \u003cem\u003eCC\u003c/em\u003e cerebral cortex, \u003cem\u003eF\u003c/em\u003e fornix, \u003cem\u003eT\u003c/em\u003e thalamus, \u003cem\u003e*\u003c/em\u003e dentate gyrus, \u003cem\u003e# \u003c/em\u003ecornu ammonis regions.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/c5277b78277fa23e6728b29a.jpeg"},{"id":92586702,"identity":"1d2d20f3-ec85-4874-9c94-c42b95eb6633","added_by":"auto","created_at":"2025-10-01 10:48:29","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":205188,"visible":true,"origin":"","legend":"\u003cp\u003eCoronal brain slices obtained from fresh, fixed, and softened brain hemispheres are shown in panels A–C. Enlarged views of the hippocampal region, indicated by red rectangular boxes in each panel, are also presented (A–C) to highlight anatomical distinctions.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCA\u003c/em\u003e cornu ammonis regions, \u003cem\u003eCC\u003c/em\u003ecorpus callosum, \u003cem\u003eIB\u003c/em\u003e infra blade, \u003cem\u003eSB\u003c/em\u003e supra blade\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/79961ba8593527a6e97582eb.jpeg"},{"id":92587638,"identity":"e36f66b1-9976-4eab-85fa-708cb080ce94","added_by":"auto","created_at":"2025-10-01 10:56:29","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":153173,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative hematoxylin and eosin (H\u0026amp;E)-stained coronal brain slices are shown for each experimental group (A–E). These images were generated by montaging multiple micrographs acquired using a 4× objective lens. Enlarged views of the boxed regions in panels A–E are presented in panels F–J, respectively, to highlight hippocampal sub-regions.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCA\u003c/em\u003e cornu ammonis regions, \u003cem\u003eFC\u003c/em\u003e fresh coronal, \u003cem\u003eFM\u003c/em\u003e fresh medial, \u003cem\u003eSM\u003c/em\u003esoftened medial, \u003cem\u003eSC \u003c/em\u003esoftened coronal.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/3d3b7040c6ad39575490c645.jpeg"},{"id":92586700,"identity":"2cd0ed7c-b0ae-4528-8ab5-0c63e6b67a55","added_by":"auto","created_at":"2025-10-01 10:48:29","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":65410,"visible":true,"origin":"","legend":"\u003cp\u003eThe surface areas (mm²) of the cornu ammonis (CA) regions across experimental groups are presented as bar graphs showing mean ± standard deviation (SD). Statistical analysis using a one-way ANOVA revealed no significant differences among the groups for each CA sub-region.\u003c/p\u003e\n\u003cp\u003eFC fresh coronal, FM fresh medial, SM softened medial, SC softened coronal.\u003c/p\u003e","description":"","filename":"image8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/6fd9e173c6a1fd2d625f4486.jpg"},{"id":97649756,"identity":"7be8a28c-d816-47f5-a8f7-5305f029a779","added_by":"auto","created_at":"2025-12-08 05:54:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2517190,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/e8eb546c-2628-49c5-a84c-35d8e5ccaf2a.pdf"},{"id":92587637,"identity":"5c2c94ab-925f-4ed9-a73e-c8b83debd626","added_by":"auto","created_at":"2025-10-01 10:56:29","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":276295,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract..jpg","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/99566f4516f1f03261492e55.jpg"},{"id":92586734,"identity":"36d0aa7c-e346-436f-a280-88de3ce42a6c","added_by":"auto","created_at":"2025-10-01 10:48:30","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":63173312,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryVideo1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/8915dd5089145932a4fb0699.mp4"},{"id":92586716,"identity":"8c4fcee8-1a9e-4050-ae4c-b79da21c65a6","added_by":"auto","created_at":"2025-10-01 10:48:29","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":33659787,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryVideo2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/7a8bd18168176501d4d57ef2.mp4"},{"id":92586732,"identity":"24f3ad59-d0a8-4e47-a1a5-6791bc29db1c","added_by":"auto","created_at":"2025-10-01 10:48:30","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":43849021,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryVideo3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/f3b9115495d9c804dbfe1119.mp4"},{"id":92587641,"identity":"b17756a5-a154-4b97-8c1a-9d06c46ad939","added_by":"auto","created_at":"2025-10-01 10:56:29","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":22339801,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryVideo4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/be1c29f8eba1f7af9c148513.mp4"},{"id":92586717,"identity":"f668c121-9bab-4380-bee5-dc3cd939c7ac","added_by":"auto","created_at":"2025-10-01 10:48:29","extension":"mp4","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":23326658,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryVideo5.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/efff18f7e882ee2e6c8e2bcb.mp4"},{"id":92586736,"identity":"d068c9ff-f20f-48f2-9a28-8ab7adf85f62","added_by":"auto","created_at":"2025-10-01 10:48:32","extension":"mp4","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":57833771,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryVideo6.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/508074eb46a3d3a1c0427b69.mp4"},{"id":92586735,"identity":"23112ec9-b965-4bdb-80e4-83a3c22ebb05","added_by":"auto","created_at":"2025-10-01 10:48:30","extension":"mp4","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":18790230,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryVideo7.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7588763/v1/44e6879e86904ec0ff87e5d5.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"Standardizing dentate gyrus isolation for molecular adult hippocampal neurogenesis studies: a comparative and tissue softening-enabled workflow","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe DG, a sub-region of the hippocampus (HP), contains the sub-granular zone, the primary site of AHN (Piatti etl al. 2013; Kempermann et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lazarov \u0026amp; Hollands 2015). AHN generates new excitatory principal neurons in an activity-dependent manner, supporting cognitive processes and being implicated in numerous neurological and psychiatric disorders (Go\u0026ccedil;alves et al. 2016; Moreno-Jim\u0026eacute;nez et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Since its discovery by Altman \u0026amp; Das (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1965\u003c/span\u003e), AHN has become a central topic in contemporary neuroscience.\u003c/p\u003e\u003cp\u003eImmunohistochemistry on sectioned brain tissue remains a cornerstone of AHN research (von Bohlen und Halbach 2011; Kempermann et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). However, deeper molecular investigations such as transcriptomic and proteomic analyses require freshly isolated DG to preserve the integrity of nucleic acids and proteins (Hagihara et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Accordingly, to preserve spatial gradients and cell-type composition for downstream molecular analyses, AHN research relies critically on precise DG procurement, free from contamination by adjacent hippocampal regions such as CA1-CA3 (Ianov et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Alkadhi \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Despite its importance, most published molecular studies lack detailed methodological descriptions or visual documentation of DG dissection, limiting reproducibility and transparency.\u003c/p\u003e\u003cp\u003eOnly a few methodological studies provide detailed guidance on DG removal (Hagihara et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gilley et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Despite this centrality, the two canonical DG microdissection strategies have never been compared head-to-head under matched conditions. Gilley et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and Guo et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) described a technique involving serial coronal sectioning and excision of DG in fragments. In contrast, Hagihara et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) introduced an approach from the medial surface of the hemisphere that permits the removal of the DG as an intact tissue. In this study, we refer to these as the coronal and medial approaches, respectively.\u003c/p\u003e\u003cp\u003eIn this context, we directly compared the two canonical DG microdissection approaches and established a practical, adoptable workflow that can be implemented using paraformaldehyde (PFA)-fixed archival brains commonly available in neuroscience laboratories. Because long-term fixed tissue is often considered suboptimal for microdissection, owing to fragility and loss of natural color/contrast needed for neuroanatomical border visibility, we employed a simple 15-day slow-running-water rinse to soften the tissue and restore landmark visibility, thereby enabling precise DG isolation from existing repositories. We then performed a side-by-side validation of the medial and coronal strategies across fresh, fixed, and softened-fixed rat hemispheres, quantifying (i) time to isolate the DG and (ii) residual CA1-CA3 area on histology after DG removal as an objective measure of anatomical specificity. By grounding the study in a direct comparison of two widely referenced methods (Hagihara et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gilley et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and incorporating a feasible softening step for archival tissue, we provide a reproducible procurement framework that enhances molecular precision and cross-study comparability in AHN research, while also reducing reliance on new animals as a secondary benefit.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEthics\u003c/h2\u003e\u003cp\u003eAll procedures complied with institutional and national guidelines and were approved by the Mersin University Ethics Committee (2016/09).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eWe used 21 adult female Sprague Dawley rats (6\u0026ndash;7 months; ~190\u0026ndash;210 g). For fixed cohorts, rats were perfused with saline followed by ice-cold 4% PFA in 0.1 M PBS. Brains were immersion-fixed for 48 h in 4% PFA, then stored 4 years at 4\u0026deg;C in 0.1\u0026ndash;0.5% PFA in PBS. Fresh hemispheres were obtained under deep ketamine/xylazine anesthesia.\u003c/p\u003e\n\u003ch3\u003eExperimental Design\u003c/h3\u003e\n\u003cp\u003eWe designed seven groups (n\u0026thinsp;=\u0026thinsp;3 hemispheres/group) were defined:\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e1) Control (no DG removal) a positive control group in the histological assessment of DG dissection success; 2) Fixed-Medial (FXM), PFA fixed hemispheres, dissected with the medial approach; 3) Fixed-Coronal (FXC), PFA fixed hemispheres, dissected with the coronal approach; 4) Softened-Medial (SM), PFA fixed hemispheres that subjected to softening procedure, dissected with the medial approach; 5) Softened-Coronal (SC), PFA fixed hemispheres that subjected to softening procedure, dissected with the coronal approach ; 6) Fresh-Medial (FM), freshly held hemispheres, dissected with the medial approach; 7) Fresh-Coronal (FC), freshly held hemispheres, dissected with the coronal approach (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eBrain Hemisphere Softening Procedure\u003c/h3\u003e\n\u003cp\u003eTo improve the texture and handling of PFA-fixed rat brain hemispheres, we leached residual fixative using a continuous, slow-running tap water rinse, verifying the flow several times daily. In pilot trials, using restoration of hippocampal color contrast and tactile pliability as endpoints, 15 days yielded the best balance; this duration was therefore adopted for the softening protocol.\u003c/p\u003e\n\u003ch3\u003eDG Dissection via Medial Approach\u003c/h3\u003e\n\u003cp\u003eWe adapted the medial side dissection protocol from Hagihara et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) (Supplementary Videos 1\u0026ndash;3, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDG Dissection via Coronal Approach\u003c/h2\u003e\u003cp\u003eWe adapted the coronal slice dissection protocol from Gilley et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) (Supplementary Videos 4\u0026ndash;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Prior to dissection, each brain hemisphere was placed on a stable platform positioned over an ice bucket, and 600 \u0026micro;m-thick coronal slices were rapidly sectioned (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter the sectioning We utilized the following steps that are also illustrated in a schematic (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eVibratome Sectioning\u003c/h3\u003e\n\u003cp\u003eWe performed vibratome sectioning either prior to applying the medial approach or following the coronal dissection, depending on the study group (as summarized in the experimental design; see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The procedure was adapted from previously published protocols (\u0026Ouml;zt\u0026uuml;rk \u0026amp; Ko\u0026ccedil; \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; \u0026Ouml;zt\u0026uuml;rk et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). We embedded brain hemispheres in 1% (w/v) agarose and sectioned into 600 \u0026micro;m thick coronal slices using a vibratome (Leica Microsystems, Buffalo Grove, IL) (see Supplementary Video 7).\u003c/p\u003e\u003cp\u003e\u003cb\u003eOutcome measures\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eOperational performance: time (seconds) to complete DG isolation from one hemisphere.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eAnatomical specificity: residual CA1-CA3 surface area on H\u0026amp;E-stained coronal sections after DG removal, quantified using the freehand selection tool in ImageJ, National Institutes of Health, Bethesda, MD; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.nih.gov/ij\u003c/span\u003e\u003cspan address=\"https://imagej.nih.gov/ij\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\n\u003ch3\u003eHematoxylin and Eosin Staining\u003c/h3\u003e\n\u003cp\u003eTo assess the area sizes of CA regions (1\u0026ndash;3) in the DG-excised brain hemispheres, we performed H\u0026amp;E staining with 600 \u0026micro;m thick coronal slices along with the experimental groups.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eImaging and Video Capturing\u003c/h2\u003e\u003cp\u003eTo demonstrate the whole, HE stained coronal slices in single piece, we montaged several adjacent images captured by (Olympus, SZ51) light microscope, equipped with an Olympus LC30 digital camera (Olympus Optical Company Ltd, Tokyo, Japan). We created and montaged the videos by using Microsoft Clipchamp application (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://clipchamp.com/en/video-editor-free\u003c/span\u003e\u003cspan address=\"https://clipchamp.com/en/video-editor-free\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e online video editor, video compressor, video converter). The original speed of the videos is accelerated by two times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eWe performed statistical analyses with the free trial version of IBM SPSS Statistics 26 software. Initially, we tested the normality and equality of variances of each data set by using Shapiro-Wilk and Levene\u0026rsquo;s tests, respectively. As we detected that the normality assumption and Levene\u0026rsquo;s test were not violated, we used independent Samples T test or one-way ANOVA for each of the normally distributed dependent variable. We presented reported values as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, t(df) and p.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eVisibility and handling in archival tissues\u003c/h2\u003e\u003cp\u003eWhen we attempted to practice the coronal (Gilley et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and medial (Hagihara et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) DG dissection approaches on 4% PFA fixed brain hemispheres with 4 years of archival age, we found that the tissues were thoroughly stiff and fragile which prevented smooth operation of dissection (Supplementary Video 2 and Video 5). We realized that due to the major color alternation on the tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C), demarcating the borders of HP from the surrounding structures and sub-HP regions were extremely difficult on the medial surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) and on the coronal slices (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), relative to the fresh brains.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eSoftening restores visibility and handling in archival tissue\u003c/h2\u003e\u003cp\u003eThe 15-day softening restored color contrast and tissue pliability, improving visibility of the hippocampal borders on both medial surfaces and coronal slices. Operational comfort increased, particularly for the coronal approach. At the 15th day of the procedure, we detected significant color change in the HP as compared to the fixed hemispheres (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). On the medial surface of the softened hemispheres, we saw that the borders of HP and surrounding structures became conveniently visible as compared to the fixed brain hemispheres (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). We further observed strong improved visibility of the neuroanatomical borders in the fixed vs softened brains in the coronal slices (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eAfter the softening procedure, we detected markedly improved operational comfort on coronal approach dissection (Supplementary Video 6), whereas the smoothness of the tissue was still not at the desired level with the medial approach (Supplementary Video 3).\u003c/p\u003e\u003cp\u003eWe obtained several measurements in the current study. Initially, we tested the normality and equality of variances of each data set by using Shapiro-Wilk and Levene\u0026rsquo;s tests, respectively. As we detected that the normality assumption and Levene\u0026rsquo;s test were not violated, we used independent Samples T test or one-way ANOVA for each of the normally distributed dependent variables.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eOperational performance: medial is faster than coronal\u003c/h2\u003e\u003cp\u003eIn the fresh and fixed softened brains, we recorded the total dissection duration to remove the entire DG from a single hemisphere both with medial and coronal approaches (for descriptive statistics, see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). On fresh hemispheres, we found that the medial dissection was significantly faster than the coronal (FM vs FC, 51.67\u0026thinsp;\u0026plusmn;\u0026thinsp;6.51 s vs 125.33\u0026thinsp;\u0026plusmn;\u0026thinsp;8.50 s; t(4)\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;11.80, p\u0026thinsp;=\u0026thinsp;0.00029). The same pattern held for softened-fixed hemispheres (SM vs SC, 301.66\u0026thinsp;\u0026plusmn;\u0026thinsp;12.34 s vs 727.33\u0026thinsp;\u0026plusmn;\u0026thinsp;16.62; t(4)\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;35.61, p\u0026thinsp;=\u0026thinsp;3.71\u0026times;10⁻⁶) (for independent T test statistics, see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As expected, both approaches were slower in softened-fixed than fresh tissue, both approaches were slower in softened-fixed than fresh tissue (FM vs SM: t(4)\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;31.01, p\u0026thinsp;=\u0026thinsp;6.43\u0026times;10⁻⁶; FC vs SC: t(4)\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;55.68, p\u0026thinsp;=\u0026thinsp;6.22\u0026times;10⁻⁷).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDescriptive statistics for DG removal duration (s)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eApproach\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal DG removal duration (s)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e51.70 (6.51)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e125.33 (8.62)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e301.66 (12.34)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e727.33 (16.62)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eTotal DG dissection durations in seconds among the groups.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cem\u003eDG\u003c/em\u003e dentate gyrus, \u003cem\u003eFC\u003c/em\u003e fresh coronal, \u003cem\u003eFM\u003c/em\u003e fresh medial, \u003cem\u003eSM\u003c/em\u003e soften medial, \u003cem\u003eSC\u003c/em\u003e softened coronal.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIndependent t-test for DG removal duration\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT(df)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ep\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e95% CI\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFM vs FC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-11.80 (4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.00029\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e\u003cp\u003e[-90.95-56.30]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM vs SC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-35.61 (4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e3.71x10\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;6\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e\u003cp\u003e[-458.85-392.47]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFM vs SM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-31.01 (4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e6.43x10\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;6\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e\u003cp\u003e[-272.34-227.59]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFC vs SC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-55.68 (4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e6.22x10\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;7\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e\u003cp\u003e[-632.01-571.98]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eBold font indicates a significant p-value of \u0026lt;\u0026thinsp;.05.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cem\u003eCI\u003c/em\u003e confidence interval, \u003cem\u003eDF\u003c/em\u003e degrees of freedom, \u003cem\u003eFC\u003c/em\u003e fresh coronal, \u003cem\u003eFM\u003c/em\u003e fresh medial, \u003cem\u003eSM\u003c/em\u003e soften medial, \u003cem\u003eSC\u003c/em\u003e softened coronal.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eAnatomical specificity: comparable across strategies and tissue states\u003c/h2\u003e\u003cp\u003eLastly, to estimate the success of DG dissection, we measured the area sizes of the CA1-3 regions on the H\u0026amp;E stained coronal slices (600 \u0026micro;m thick) taken after the DG removal procedures. We placed the measurements in the following groups; Control (no DG removal, intact brain hemispheres as regional control) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA); FM (fresh medial approach) (freshly obtained hemispheres, dissected with medial approach) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), FC (fresh coronal approach) (freshly obtained hemispheres, dissected with coronal approach) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), SM (softened medial approach) (paraformaldehyde fixed hemispheres that subjected to softening procedure, dissected with medial approach) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), SC (softened coronal approach) (paraformaldehyde fixed hemispheres that subjected to softening procedure, dissected with coronal approach) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe found that residual CA1-CA3 areas did not differ significantly across groups (one-way ANOVA, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; see Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), indicating comparable anatomical specificity for medial and coronal strategies whether performed on fresh or softened-fixed tissue.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDescriptive statistics of CA1-3 area sizes\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eMean (sd)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCA1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.27 (0.023)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.23 (0.02)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.25 (0.049)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.16 (0.02)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.29 (0.03)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCA2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.032 (0.002)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.023 (0.002)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.027 (0.008)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.025 (0.01)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.036 (0.002)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCA3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.10 (0.014)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.076 (0.008)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.11 (0.026)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.091 (0.037)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.11 (0.017)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eCA\u003c/em\u003e cornu ammonis, \u003cem\u003eFC\u003c/em\u003e fresh coronal, \u003cem\u003eFM\u003c/em\u003e fresh medial, \u003cem\u003eSD\u003c/em\u003e standard deviation, \u003cem\u003eSM\u003c/em\u003e soften medial, \u003cem\u003eSC\u003c/em\u003e softened coronal.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOne-way ANOVAs\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF (df,error)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ep\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCA1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.36 (4,10)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.123\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCA2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.72 (4,10)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.597\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCA3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.642 (4,10)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.644\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003cem\u003eF\u003c/em\u003e Indicates the statistic for ANOVA is the ratio of the mean square for the between groups divided by the mean square within groups.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003cem\u003eCA\u003c/em\u003e cornu ammonis, \u003cem\u003eDF\u003c/em\u003e degrees of freedom.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides a head to head validation of two canonical DG microdissection strategies, demonstrating that the medial approach is faster while both medial and coronal approaches achieve comparable anatomical specificity as quantified by residual CA1-CA3 area. By isolating the dissection strategy as the key pre-analytical variable, our results address a long-standing gap that directly impacts the reproducibility and comparability of AHN experiments that depend on precisely procured DG tissue.\u003c/p\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eMedial vs coronal: visibility vs speed\u003c/h2\u003e\u003cp\u003eIn fresh brains, natural color contrast and tissue pliability made both strategies smooth to execute; nonetheless, the medial approach was consistently faster (~\u0026thinsp;52\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5 s) than the coronal approach (~\u0026thinsp;125\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5 s), with high operator comfort for both. By contrast, PFA-fixed tissue was stiff, fragile, and low-contrast, obscuring DG-CA borders and reducing comfort regardless of strategy, an observation consistent with prior emphasis on maintaining clear DG-CA boundaries (Hagihara et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). To mitigate this, a 15-day slow-running-water rinse improved pliability and border visibility. After softening, coronal dissections benefitted the most, with handling and visibility approaching the fresh tissue experience; the medial approach remained workable but was slightly less comfortable than in fresh tissue due to its reliance on precise medial surface landmarks during diencephalon removal. As expected, both strategies took longer in softened-fixed than in fresh tissue, but the relative ranking in speed was preserved (medial\u0026thinsp;\u0026lt;\u0026thinsp;coronal), while anatomical specificity remained comparable across approaches. Taken together, we recommend choosing medial when speed and intact-block removal are priorities, and coronal when maximal border visibility and stepwise control are preferred, especially on softened archival tissue.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3R-aligned training and workflow standardization\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe 15-day softening protocol provides an excellent operational surrogate for DG microdissection, readily adoptable by many neuroscience labs to standardize technique and workflow for DG and other hard to dissect neuroanatomical regions. This enables training and protocol optimization without excessive new animal use. In line with the 3Rs framework (Replacement, Reduction, Refinement) as articulated by Russell and Burch (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1959\u003c/span\u003e) (Russell and Burch \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1959\u003c/span\u003e), this surrogate replaces portions of training that would otherwise require newly sacrificed animals, reduces overall numbers for skill acquisition/pilot work, and refines procedures via improved border visibility and handling.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eImplications for AHN workflows\u003c/h2\u003e\u003cp\u003eFor AHN studies, precise DG procurement is critical because the DG shows robust dorsal-ventral molecular and epigenetic differentiation (Christensen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lothmann et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), along with cell and circuit level differences including divergent mossy cell projections and synaptic modulation along the axis (Houseret et al. 2021; Trompoukis \u0026amp; Papatheodoropoulos \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Variation in dissection boundaries or procedural time can therefore plausibly bias downstream molecular readouts and histological quantification. Our head to head comparison indicates that strategy choice affects speed and operational comfort without compromising anatomical specificity. Although we did not assay molecular endpoints, favoring a medial microdissection when minimizing \u003cem\u003eex vivo\u003c/em\u003e time is the priority and a coronal microdissection when maximal boundary visibility or precise dorso-ventral targeting is required is a logical way to reduce pre-analytical variability in AHN pipelines. This recommendation is motivated by robust dorso-ventral differences in the dentate gyrus (Christensen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lothmann et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Houseret et al. 2021; Trompoukis \u0026amp; Papatheodoropoulos \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRegarding the degradation of nucleic acids and proteins, preferring one of these approaches over another when performed on fresh tissues could be a choice. The storage temperature and duration have an impact on the RNA integrity of brain tissue sections which might be the most critical factors affecting gene expression level in RNA sequencing (Jia et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, Catts and colleagues detected no significant change on RNA yield, RNA purity and 28S/18S optical density at the 6th postmortem hour of the mouse brain tissue (Catts et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Therefore, we expect that the approximately one minute longer duration of the coronal approach would not significantly impact RNA quality.\u003c/p\u003e\u003cp\u003eWe summarize our study experience to guide researchers; detailed notes are provided in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOperational comparison of DG microdissection strategies across tissue states and recommended use-cases\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTissue state\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eApproach\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSpeed (relative)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOperator comfort \u0026amp; border visibility\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAnatomical specificity (residual CA1\u0026ndash;CA3)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRecommended use-case\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNotes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedial\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFaster (e.g., ~\u0026thinsp;52\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5 s)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh comfort; intact-block removal feasible\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eComparable to coronal (ANOVA n.s.)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMinimize ex-vivo time; rapid procurement for RNA/protein-sensitive workflows\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRequires confident medial-surface landmarking during diencephalon removal\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCoronal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSlower (e.g., ~\u0026thinsp;125\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5 s)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eExcellent slice-face visibility; stepwise control\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eComparable to medial approach (ANOVA n.s.)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMaximal border control; dorsal-ventral sampling strategies\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSlightly longer procedure; maintain strict cold chain\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoftened-fixed (15-day rinse)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedial\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSlower than fresh; faster than coronal (rank preserved)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWorkable, but comfort lower than fresh due to reliance on medial landmarks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eComparable to coronal (ANOVA n.s.)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTraining/standardization without new animals; pilot histology\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOperational surrogate; not for molecular assays Only for practicing\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoftened-fixed (15-day rinse)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCoronal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSlower than fresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMarkedly improved visibility; handling approaches fresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eComparable to medial (ANOVA n.s.)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTraining; optimizing border definitions; complex borders\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eBest visibility on softened tissue; good for stepwise teaching\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFixed (unsoftened)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedial/\u003c/p\u003e\u003cp\u003eCoronal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSlowest\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLow visibility; stiff/fragile, difficult borders\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNot evaluated for advantage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNot recommended; soften first\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eApply 15-day slow-running-water rinse before use\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eSpeed entries highlight the relative rank; example means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD are shown for fresh tissue from the present dataset. For softened-fixed tissue, both strategies were slower than fresh, but the medial\u0026thinsp;\u0026lt;\u0026thinsp;coronal speed ranking persisted. Summary of operational performance and anatomical fidelity for medial vs coronal DG microdissection across fresh, softened-fixed, and fixed (unsoftened) tissue states. The medial approach is faster; coronal provides superior visibility, especially after softening. Anatomical specificity is comparable between strategies (ANOVA n.s.) across states. \u003cem\u003eDG\u003c/em\u003e dentate gyrus, \u003cem\u003eAHN\u003c/em\u003e adult hippocampal neurogenesis, \u003cem\u003en.s.\u003c/em\u003e not significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eLimitations\u003c/h2\u003e\u003cp\u003eSample sizes per group were modest (n\u0026thinsp;=\u0026thinsp;3), and we did not quantify RNA/protein integrity post-procurement from archival tissue here. Future studies should pair this workflow with RNA/DNA/protein quality control metrics (e.g. RIN and DIN) and extend validation to mouse models and single-nucleus preparations for AHN focused omics.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe provide a validated, adoptable workflow for DG microdissection that (i) compares medial vs coronal strategies under matched conditions, (ii) demonstrates comparable anatomical specificity across strategies and tissue states, (iii) shows that a 15-day softening rescues archival PFA-fixed brains for precise DG procurement, and (iv) enables molecularly precise AHN studies while limiting additional animal use.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was carried out at the Neuroanatomy and Experimental Research Laboratory, which was established with the infrastructure grant support of the Research Projects Unit of Mersin University (Grant No. 2018-1-AP5-2895, 2020-1-AP5-4104). Consumables used in this study were partially used from the grants supported by research grant (116S458) supported by The Scientific and Technological Research Institution of T\u0026uuml;rkiye.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no conflict of interest. All authors read and approved the final manuscript and attest to the integrity of the original data and the analysis reported in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study has been approved by Ethics Committee of Mersin University (2016/09).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data (accessible part) presented in this study is available upon reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u003c/strong\u003e [Turan Ko\u0026ccedil;, Nail Can \u0026Ouml;zt\u0026uuml;rk]; \u003cstrong\u003eMethodology:\u003c/strong\u003e [Turan Ko\u0026ccedil;, Nail Can \u0026Ouml;zt\u0026uuml;rk]; \u003cstrong\u003eFormal analysis and investigation:\u003c/strong\u003e [Turan Ko\u0026ccedil;, Nail Can \u0026Ouml;zt\u0026uuml;rk]; \u003cstrong\u003eWriting - original draft preparation:\u003c/strong\u003e [Turan Ko\u0026ccedil;, Nail Can \u0026Ouml;zt\u0026uuml;rk]; \u003cstrong\u003eWriting - review and editing:\u0026nbsp;\u003c/strong\u003e[Turan Ko\u0026ccedil;, Nail Can \u0026Ouml;zt\u0026uuml;rk]; \u003cstrong\u003eFunding acquisition:\u003c/strong\u003e [Nail Can \u0026Ouml;zt\u0026uuml;rk]; \u003cstrong\u003eResources:\u003c/strong\u003e [Nail Can \u0026Ouml;zt\u0026uuml;rk]; \u003cstrong\u003eSupervision\u003c/strong\u003e: [Nail Can \u0026Ouml;zt\u0026uuml;rk]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the use of artificial intelligence-assisted language tools (ChatGPT, Open AI) exclusively for the purpose of enhancing grammar, articulation, and scientific tone. These tools were not used for content generation, data analysis, data interpretation, or any other aspect of scientific authorship. All intellectual content was developed solely by the authors, and the final manuscript was critically reviewed and approved by all contributors. All Supplementary Videos include embedded captions and descriptions.\u003c/p\u003e\n\u003cp\u003eThe authors also extend their sincere gratitude to Prof. Zeliha Kurtoğlu Olgunus (former Head of the Department of Anatomy) as well as to the members of the Department of Anatomy, Mersin University Faculty of Medicine, for their contributions to the development of the Neuroanatomy and Experimental Research Laboratory (Project No. 2018-1-AP5-2895).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlkadhi KA (2019) Cellular and Molecular Differences Between Area CA1 and the Dentate Gyrus of the Hippocampus. 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Nat Commun 9(1):298. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-017-02748-x\u003c/span\u003e\u003cspan address=\"10.1038/s41467-017-02748-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"dentate gyrus, adult hippocampal neurogenesis, microdissection, tissue softening","lastPublishedDoi":"10.21203/rs.3.rs-7588763/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7588763/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdult hippocampal neurogenesis (AHN) studies depend on dentate gyrus (DG) tissue isolated with high anatomical fidelity and controlled pre-analytical steps. To our knowledge, the two widely used DG microdissection approaches, medial (intact-block) and coronal (slice-guided) have not been directly compared under matched conditions, and the value of a simple tissue-softening step for operational standardization has not been quantified.\u003c/p\u003e\u003cp\u003eTo provide a comparative, quantitative validation of the medial and coronal DG microdissection approaches and to establish a tissue softening enabled workflow that laboratories can adopt for training and standardization.\u003c/p\u003e\u003cp\u003eAdult rat hemispheres were assigned to seven groups (n\u0026thinsp;=\u0026thinsp;3 hemispheres/group): fresh-medial, fresh-coronal, fixed-medial, fixed-coronal, softened-medial, softened-coronal, and intact control (fixed). A 15-day slow running-water rinse softened archival tissue. Outcomes were (i) operational performance (time to isolate DG) and (ii) anatomical specificity (residual CA1-CA3 area on H\u0026amp;E after the DG removal).\u003c/p\u003e\u003cp\u003eIn fresh tissue, the medial approach isolated DG in 51.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5 vs 125.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5 s for the coronal approach (~\u0026thinsp;2.4\u0026times; difference). In softened fixed tissue, both approaches were slower, but the speed ranking (medial\u0026thinsp;\u0026lt;\u0026thinsp;coronal) was preserved. Anatomical specificity did not differ among groups (residual CA1-CA3; one-way ANOVA, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Softening improved border visibility and handling, particularly for the coronal approach, providing an operational surrogate for training and standardization.\u003c/p\u003e\u003cp\u003eUnder matched conditions, medial approach offers faster procurement while coronal maximizes border visibility; both maintain comparable anatomical specificity. The 15-day softening protocol supports 3R-aligned training and standardization.\u003c/p\u003e","manuscriptTitle":"Standardizing dentate gyrus isolation for molecular adult hippocampal neurogenesis studies: a comparative and tissue softening-enabled workflow","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-01 10:48:23","doi":"10.21203/rs.3.rs-7588763/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":"91e84b65-8bce-41c1-8650-75cad7fdb4bb","owner":[],"postedDate":"October 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T05:53:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-01 10:48:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7588763","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7588763","identity":"rs-7588763","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}