Fluorophore-Conjugated Antibodies Enable In Vitro Mapping of GLP-1 and GIP in Rat Small Intestine

Fluorophore-Conjugated Antibodies Enable In Vitro Mapping of GLP-1 and GIP in Rat Small Intestine | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Fluorophore-Conjugated Antibodies Enable In Vitro Mapping of GLP-1 and GIP in Rat Small Intestine Chin Hong Lim, Ciaran Lim, Koy Min Chue, Nan Guang Tan, Baldwin Yeung, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9251347/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Bariatric surgery provides substantial weight loss and metabolic improvements in severe obesity. RYGB, DS, OAGB, and SADI-S share a mechanism of diverting nutrient flow away from the duodenum and proximal jejunum, altering gut hormone signalling and incretin responses. However, bypass length is guided largely by surgeon experience and retrospective evidence, limiting precision due to variability in individual small-bowel anatomy. Target-specific visualization of enteroendocrine and absorptive markers could enable more precise bypass design. We evaluated whether conjugated fluorophore-labelled antibodies against GLP-1, GIP, and SGLT-1 can be detected in vitro in rat small-intestinal tissue as a prerequisite for subsequent in vivo studies. Male 12-week-old Wistar rats were euthanized for intestinal segment collection; monoclonal antibodies were conjugated with IRDye 800CW or Alexa Fluor 488 and applied to formalin-fixed, paraffin-embedded small-bowel sections using immunofluorescence. Fluorescein–anti GLP-1 showed uptake localized to the terminal ileum, while IR800CW–anti GIP localized to the proximal small bowel, with signal observed in enterocytes and in lamina propria vasculature and subserosal layers. In contrast, MB633–anti SGLT-1 showed no detectable uptake across tested segments or dilutions (1:100–1:400), potentially due to limited epitope accessibility, altered SGLT-1 expression, steric effects, and/or species or isoform mismatch. Conjugated antibodies enabled segment-specific in vitro detection of GLP-1 and GIP but not SGLT-1. These results support the feasibility of fluorophore-labelled incretin-marker imaging (Cellvizio or fluorescence laparoscopy) for future intra-operative mapping, with the long-term goal of refining personalized bypass design while minimizing malnutrition risk. Health sciences/Diseases Health sciences/Gastroenterology Health sciences/Medical research Bariatric surgery GLP-1 GIP SGLT-1 Immunofluorescence Fluorescent antibodies Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Bariatric surgery has emerged as a crucial intervention for severe obesity and its associated metabolic disorders, providing substantial weight loss and improvements in conditions such as type 2 diabetes, hypertension, and dyslipidaemia [ 1 , 2 ]. Among the major surgical approaches-Roux-en-Y gastric bypass (RYGB), duodenal switch (DS), one-anastomosis gastric bypass (OAGB), and single-anastomosis duodeno-ileal bypass plus sleeve gastrectomy (SADI-S) a shared underlying mechanism is the diversion of food away from the duodenum and proximal jejunum. This change in gastrointestinal routing alters gut physiology and promotes metabolic benefits. Two main explanatory frameworks are commonly discussed: the foregut hypothesis and the hindgut hypothesis. The foregut hypothesis proposes that bypassing the duodenum and proximal jejunum reduces nutrient absorption while also modulating gut hormone secretion. By limiting nutrient exposure to the upper intestine, this approach is associated with reduced ghrelin secretion, a hormone that stimulates appetite. In contrast, the hindgut hypothesis emphasizes the rapid delivery of nutrients to the distal small intestine, particularly the ileum and colon [ 3 , 4 ]. This promotes increased secretion of incretin hormones, including glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and peptide YY (PYY), which are central to the regulation of glucose metabolism and appetite. GLP-1 has received considerable attention due to its diverse metabolic effects. It enhances insulin secretion, suppresses glucagon release, slows gastric emptying, and increases satiety, thereby supporting weight loss and improved glycaemic control [ 5 ]. Likewise, GIP and PYY contribute to appetite regulation and energy homeostasis [ 6 , 7 ]. By redirecting nutrient flow toward the distal small bowel, bariatric procedures may strengthen these hormonal pathways, increasing the incretin response and supporting metabolic health. Despite these benefits, a major trade-off remains. Bypassing excessive lengths of the small bowel can result in macronutrient and micronutrient deficiencies due to reduced absorptive length and surface area [ 8 ]. This creates an ongoing clinical challenge for surgeons: optimizing weight loss and metabolic improvement while minimizing malnutrition risk. At present, selecting an appropriate bypass length often depends largely on surgeon experience and retrospective clinical evidence, which remains limited in scientific robustness. Moreover, the methods used to measure bypass length are relatively basic. Distances are commonly estimated using tape or ruler measurements, or laparoscopic instruments, from fixed anatomical landmarks such as the duodenojejunal (DJ) flexure or ileocecal valve, using stepwise hand-over-hand techniques (Fig. 1 ). However, this approach is vulnerable to variability because adult small bowel length differs substantially between individuals. As a result, it remains difficult to develop universally applicable guidelines for bypass length. These limitations highlight the need for precision medicine in bariatric surgery. By incorporating individual anatomical and physiological characteristics, surgical strategies may be tailored to maximize metabolic benefit while reducing the likelihood of nutritional deficiencies. Progress in understanding the role of L-cells in incretin hormone secretion, together with advances in imaging, could enable more individualized surgical interventions. Ultimately, achieving the optimal balance in bypass length may improve patient outcomes and reduce long-term complications. The primary objective of this study is to evaluate the efficacy of conjugated fluorophore-labelled antibodies for GLP-1, GIP, and SGLT-1 in targeted tissue in vitro, as a prerequisite to subsequent in vivo studies. Results There was clear evidence of absolute uptake of fluorescein–anti GLP‑1 in the terminal ileum and of IR800CW–anti GIP in the proximal small bowel (Figs. 2 & 3 ). In both cases, the fluorescence signal was localized to enterocytes as well as to capillaries within the lamina propria and the subserosal layer. Fluorescence imaging was performed using an inverted microscope (DMI6000B; Leica Biosystems) equipped with an NIR illumination source (X‑Cite 200DC; Excelitas Technologies) and an NIR filter set comprising band‑pass filters (850–890 nm) and a long‑pass emission filter (HQ800795LP; Chroma Technology Corp.). Images were acquired with a monochrome DFC365 FX camera (one 4‑million‑pixel charge‑coupled device; Leica Biosystems GmbH) and analysed using LAS‑X software (Leica Biosystems GmbH). For the fluorescein‑conjugated antibody, excitation/emission (Ex/Em) maxima were observed at 495/515 nm at a dilution of 1:200. For the IR800CW‑conjugated antibody, Ex/Em maxima were observed at 773/792 nm when used at a dilution of 1:400. In contrast, we did not detect uptake of MB633–anti SGLT‑1 in any segment of the murine small intestine, even after testing multiple antibody dilutions (1:100, 1:200, and 1:400), with an Ex/Em profile of 630/650 nm. SGLT‑1 is an essential transporter involved in intestinal glucose absorption, primarily localized to the brush border of enterocytes in the duodenum and jejunum. In the intestine, SGLT‑1 mediates glucose transport from the intestinal lumen into epithelial cells, using the sodium gradient generated by the Na⁺/K⁺ ATPase pump [ 9 ]. This glucose transport mechanism is important for efficient nutrient uptake following meals. The most likely explanation for the poor uptake of MB633–anti SGLT‑1 is inefficient antibody binding to SGLT‑1 in the small intestine, which may reflect low or altered SGLT‑1 expression and/or reduced epitope accessibility on the tissue surface. Additionally, the MB633 conjugation may impair antibody binding through steric hindrance or an unfavourable epitope–dye orientation. Other contributing factors may include species/isoform mismatch or luminal interference (e.g., mucus or digestive components) that could further limit effective antibody binding. Discussion Small-bowel physiological changes after bariatric surgery reflects that the small intestine is both an absorptive organ and an enteroendocrine signalling system, with function determined by where nutrients are delivered and when they encounter digestive secretions and hormone-secreting cells. Bariatric procedures reconfigure nutrient transit, the timing of mixing between food, bile and pancreatic enzymes, and the luminal environment that shapes both absorption and gut-hormone responses. Ngo et al. reported that anatomical variation in small-bowel length is significantly associated with glycaemic control and metabolic status [9], helping explain why outcomes differ between patients even after the same operation. In RYGB, bypass of the duodenum and proximal jejunum reduces proximal nutrient exposure while accelerating distal delivery of carbohydrates and fats, which enhances distal enteroendocrine stimulation and GLP‑1 mediated effects that improve appetite regulation and glucose homeostasis; delayed mixing can also transiently impair fat digestion and micelle formation. In DS/BPD‑DS, these effects are amplified by greater separation of nutrient flow from bile/pancreatic enzymes and a shorter common channel, increasing the likelihood of clinically meaningful malabsorption, especially fats and fat-soluble vitamins, and in severe cases protein. Sleeve-plus procedures are typically less malabsorptive than DS/BPD‑DS, but altered gastric emptying and bile acid–nutrient dynamics can still shift incretin profiles and promote micronutrient deficiencies depending on the specific “plus” anatomy [10]. Major pitfalls arise when intake and supplementation do not match the post-surgical absorptive capacity. Severe protein deficiency may result from inadequate protein intake (low dietary protein, supplement intolerance, vomiting, poor adherence) and from reduced effective absorption due to anatomy or complications such as bacterial overgrowth. In DS, the shortened absorptive window increases vulnerability to hypoalbuminemia, sarcopenia, and systemic consequences of protein-calorie malnutrition, particularly when chronic diarrhoea, surgical stress, or SIBO further impair nutrient handling. Micronutrient deficiencies follow predictable mechanistic patterns: iron and B-vitamins can decline with reduced exposure to proximal absorptive regions and altered digestion/acid conditions; calcium and vitamin D may deteriorate when absorption is less efficient; and fat-soluble vitamins (A, D, E, K) are especially at risk when fat digestion/absorption is impaired by mixing delay or common-channel shortening [11]. Across bypass configurations, SIBO can worsen both macronutrient and micronutrient depletion by promoting luminal overgrowth and impaired digestion. Variation in outcomes is therefore driven not only by procedure type, but also by the interaction of anatomy, small-bowel morphology, microbiome, and adaptive capacity over time. Limb lengths and surgical technique determine common-channel length and the site where nutrients meet bile and pancreatic enzymes, while adherence and tolerance determine nutrient delivery. Microbiome shifts and bile acid changes further modulate nutrient processing, barrier function, and hormone signalling, and complications such as SIBO, strictures, or chronic diarrhoea can alter effective absorption and symptom profiles even after technically successful surgery. Precision medicine can be strengthened by mapping the location and density of SGLT‑1, GLP‑1 secreting cells (L cells), and GIP-secreting cells (K cells). Because SGLT‑1 mediates enterocyte glucose uptake, segment-specific glucose delivery after surgery may help predict absorption efficiency, glycaemic patterns, and dietary tolerance. Distal nutrient delivery generally enhances GLP‑1 signalling, supporting satiety and improved insulin secretion, whereas K cells are more prominent proximally; thus, foregut exclusion in RYGB can reduce proximal K-cell nutrient exposure and shift incretin balance. This spatial framework can support prevention by anticipating which nutrients will contact impaired absorptive/endocrine regions, tailoring protein/carbohydrate strategies, intensifying early monitoring of high-risk micronutrients, and evaluating/treating SIBO when luminal clearance is likely impaired. In summary, identifying the distribution and density of GLP‑1, GIP, and SGLT‑1 could guide bypass length selection in RYGB, DS/BPD‑DS, and sleeve-plus to optimize metabolic benefits while minimizing malabsorption. Moving forward, we plan in vivo real-time fluorescent imaging with Cellvizio to map the intra-operative distribution and density of GLP‑1, GIP, and SGLT‑1 in rats’ small intestine and use this information to refine bypass design to prevent nutrient malabsorption [12]. Methods 1. Animal Preparation Male 12-week-old Wistar rats were housed in a barrier facility on high-efficiency particulate air (HEPA)-filtered racks in a controlled room maintained at 22 °C under a 12-h light/dark cycle (lights on at 07:00 and off at 19:00). Animals were fed autoclaved laboratory rodent diet (Teckland LM-485; Western Research Products, Laramie, WY). In this in vivo study, rats were humanely sacrificed for the collection of small intestine tissue samples. As the experimental procedure did not involve any surgical intervention or invasive manipulation prior to tissue harvesting, anaesthesia was not required. Animals were euthanized using cervical dislocation to ensure rapid and humane death. Immediately after euthanasia, the abdominal cavity was opened and the small intestine was carefully removed, rinsed in cold phosphate-buffered saline (PBS) to remove residual luminal contents, and segmented for subsequent analysis. 2. Ethical Considerations and Animal Welfare All experimental protocols were reviewed and approved by the SingHealth Institutional Animal Care and Use Committee (IACUC), and all procedures were conducted in accordance with institutional and national ethical approvals. Prior to study initiation, the experimental design was reviewed to confirm scientific necessity and the absence of viable alternatives. Throughout the study, animal welfare was prioritized by minimizing discomfort, providing appropriate housing and nutrition, and ensuring veterinary monitoring. Personnel were trained in humane handling and animal care to support ethical conduct while enabling scientific objectives. 3. Antibodies Conjugation Monoclonal antibodies specific for GLP-1, GIP, and SGLT-1 were obtained from Thermo Fisher Scientific (Waltham, MA). Antibodies were conjugated with either the IRDye 800CW or AlexaFluor 488 Protein Labelling Kit (Molecular Probes, Grand Island, NY) according to the manufacturer’s instructions [13]. Briefly, antibodies were reconstituted at 1 mg/mL in 0.1 M sodium bicarbonate. One hundred microliters of the antibody solution were added to the reactive dye and incubated for 1 hour at room temperature. Conjugated antibodies were then separated from unreacted dye using a gravity purification column. Antibody and dye concentrations in the final preparations were determined by spectrophotometric absorbance measurements ( Figure 4 ). 5. Immunofluorescence Staining of Formalin-Fixed Paraffin-Embedded Tissue Sections In vitro testing using target tissue sections involves preparing different segments of the small intestine- proximal, mid, and distal that express GLP-1, GIP, and SGLT-1. Slides were deparaffinised through sequential xylene and ethanol washes using Coplin jars. Sections were incubated in xylene for 9 minutes and then in fresh xylene for an additional 9 minutes. Dehydration was performed using a graded ethanol series: 100% ethanol for 5 minutes (twice), 95% ethanol for 5 minutes (twice), 85% ethanol for 5 minutes (twice), and 75% ethanol for 5 minutes (twice). Following dehydration, slides were washed three times in water (5 minutes per wash). After the final water wash, slides were kept hydrated until antigen retrieval was performed; drying at any point was avoided because it increased non-specific antibody binding and elevated background staining. For antigen retrieval, tissue sections were encircled with a PAP pen to localize reagents to the section. A working solution of proteinase K was prepared by diluting proteinase K in TE buffer (pH 8.0) containing 0.5% Triton-X at a 1000× dilution. Three hundred microliters of the working solution were applied to each section and incubated for 15 minutes at 37 °C. Slides were then washed with TBST (400 µL per wash) three times for 5 minutes each. For antibody incubation, slides were blocked with 400 µL of blocking buffer (1× TBST + 2% BSA) for 1–2 hours. Blocking buffer was removed, and fluorescence-conjugated primary antibodies were applied at the indicated dilutions (Fluorescein–anti GLP1 1:200; IR800CW–anti GIP 1:400; MB633–anti SGLT-1 tested at 1:100, 1:200, or 1:400). Sections were incubated with 300 µL of primary antibodies overnight at 4 °C. Primary antibodies were then removed and slides were washed three times with TBST (400 µL per wash) for 5 minutes each. After the final wash, excess TBST was removed by gently dabbing the slides dry, and antifade mounting media (2-3 drops) was applied prior to placing glass coverslips ( Figure 5 ). Fluorescence imaging is utilized to visualize the binding of the conjugated antibodies, capturing images to assess localization and intensity. Declarations Ethics Approval Statement This study involving animal subjects has been conducted in accordance with ethical guidelines. Written informed consent and approval from the Institutional Review Board (IRB) was deemed unnecessary given the nature of the study. All procedures were performed in compliance with relevant regulations to ensure the welfare of the animals involved. Data availability statement: Raw data and analysis code are available from the corresponding author upon reasonable request. Author Contribution Statement: LCH, BY and LJL conceived the experiments. LCH, CL, CKM, TNG and LO conducted the experiments. LCH, CWH, RA and JT analysed the results. LCH wrote the manuscript. Declaration of competing interest: The authors declare that they have no competing interests. Funding Declaration: The author(s) received no financial support for the research, authorship, and/or publication of this article. References Sjostrom, L. et al. Effects of Bariatric Surgery on Mortality in Swedish Obese Subjects. NEJM 357 , 741–752 (2007). Schauer, P. et al. Bariatric surgery versus intensive medical therapy for diabetes- 5-year outcomes. N Eng. J. Med. 376 , 641–651 (2017). Hickey, M. S. et al. A new paradigm for type 2 diabetes mellitus: could it be a disease of the foregut? Ann. Surg. 227 , 637–643 (1998). Rubino, F. et al. The mechanism of diabetes control after gastrointestinal bypass surgery reveals a role of the proximal small intestine in the pathophysiology of type 2 diabetes. Ann. Surg. 244 , 741–749 (2006). Holst, J. J. The physiology of glucagon-like peptide 1. Physiol. Rev. 87 (4), 1409–1439. 10.1152/physrev.00034.2006 (2007). Seino, Y., Fukushima, M. & Yabe, D. GIP and GLP-1, the two incretin hormones: Similarities and differences. J. Diabetes Investig . 1 (1–2), 8–23 (2010). Karra, E., Chandarana, K. & Batterham, R. L. The role of peptide YY in appetite regulation and obesity. J. Physiol. 587 (1), 19–25 (2009). Syn, N. L. et al. Associations of Bariatric Interventions With Micronutrient and Endocrine Disturbances. JAMA Open. 3 (6), e205123 (2020). Ngo, C. et al. Association of Total Small Bowel Length with Glycaemic Control and Metabolic Syndrome in Metabolic and Bariatric Surgery Patients: A Cross-Sectional Study in a Taiwanese Cohort. Obes. Surg. https://doi.org/10.1007/s11695-026-08594-5 (2026). Kurahashi, Y., Hojo, Y. & Shinohara, H. Sleeve Plus Procedures: A Review of Technical Evolution Along a Schematic Diagram. Obes. Surg. 35 , 4806–4813 (2025). Lewis, C. A. et al. Monitoring for micronutrient deficiency after bariatric surgery—what is the risk? Eur. J. Clin. Nutr. 77 , 1071–1083 (2023). Pohl, H. et al. Miniprobe confocal laser microscopy for the detection of invisible neoplasia in patients with Barrett's oesophagus. Gut 57 , 1648–1653 (2008). Zhao, M., Li, N. & Zhou, H. SGLT1: A Potential Drug Target for Cardiovascular Disease. Drug Des. Devel Ther. 17 , 2011–2023 (2023). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 01 May, 2026 Editor assigned by journal 01 May, 2026 Editor invited by journal 29 Apr, 2026 Submission checks completed at journal 11 Apr, 2026 First submitted to journal 11 Apr, 2026 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9251347","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":633712286,"identity":"a7044a31-e5fe-496f-8441-c9861a458ab5","order_by":0,"name":"Chin Hong 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09:10:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9251347/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9251347/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108975753,"identity":"c9fb197e-bbf3-4fb8-a25b-8fbbbce41639","added_by":"auto","created_at":"2026-05-11 10:57:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":259077,"visible":true,"origin":"","legend":"\u003cp\u003eLaparoscopic measurement of bypassed small intestinal length using marking on the instrument.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9251347/v1/e9e170e1076ddf2ddc8a6385.jpg"},{"id":108975869,"identity":"1f516834-50ec-45c7-bfa4-3f149e7d4cfb","added_by":"auto","created_at":"2026-05-11 10:58:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":719090,"visible":true,"origin":"","legend":"\u003cp\u003eRat small intestine distal ileum sections were stained with FITC-conjugated anti–GLP‑1 antibody (10× magnification).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9251347/v1/f879efdb0f709f46b23ed8fa.jpg"},{"id":108975918,"identity":"756ba85c-b163-4119-ba7b-abe18e285e1b","added_by":"auto","created_at":"2026-05-11 10:58:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":545789,"visible":true,"origin":"","legend":"\u003cp\u003eRat duodenum and proximal jejunum were stained with IR800CW–anti GIP at 10× and 20× magnification. Signals were detected using a FITC‑conjugated anti‑rabbit secondary antibody.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9251347/v1/4e7a3dddee06bf8fd202b03c.jpg"},{"id":108975915,"identity":"9cc62ae4-ba2a-4199-8d10-58b5d6c229be","added_by":"auto","created_at":"2026-05-11 10:58:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":151453,"visible":true,"origin":"","legend":"\u003cp\u003eGLP‑1–, GIP–, and SGLT‑1–specific monoclonal antibodies were fluorescently labelled with distinct fluorophores (different colours).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9251347/v1/4dabc2e84201393c8246d5c4.jpg"},{"id":108976008,"identity":"3db80e69-9acc-433b-839e-721a645f6d20","added_by":"auto","created_at":"2026-05-11 10:58:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3203819,"visible":true,"origin":"","legend":"\u003cp\u003eFormalin-fixed paraffin-embedded small-bowel segments were placed in a circular arrangement, with proximal regions at the start and distal regions at the end.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9251347/v1/f7494e8b202814069ccd0467.png"},{"id":108980306,"identity":"7d01eed0-e657-42ed-bb0a-f2335a81e1f0","added_by":"auto","created_at":"2026-05-11 12:04:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6044761,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9251347/v1/9bca8da7-6457-4f0a-a377-8504d18347e5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fluorophore-Conjugated Antibodies Enable In Vitro Mapping of GLP-1 and GIP in Rat Small Intestine","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBariatric surgery has emerged as a crucial intervention for severe obesity and its associated metabolic disorders, providing substantial weight loss and improvements in conditions such as type 2 diabetes, hypertension, and dyslipidaemia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among the major surgical approaches-Roux-en-Y gastric bypass (RYGB), duodenal switch (DS), one-anastomosis gastric bypass (OAGB), and single-anastomosis duodeno-ileal bypass plus sleeve gastrectomy (SADI-S) a shared underlying mechanism is the diversion of food away from the duodenum and proximal jejunum. This change in gastrointestinal routing alters gut physiology and promotes metabolic benefits. Two main explanatory frameworks are commonly discussed: the foregut hypothesis and the hindgut hypothesis.\u003c/p\u003e \u003cp\u003eThe foregut hypothesis proposes that bypassing the duodenum and proximal jejunum reduces nutrient absorption while also modulating gut hormone secretion. By limiting nutrient exposure to the upper intestine, this approach is associated with reduced ghrelin secretion, a hormone that stimulates appetite. In contrast, the hindgut hypothesis emphasizes the rapid delivery of nutrients to the distal small intestine, particularly the ileum and colon [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This promotes increased secretion of incretin hormones, including glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and peptide YY (PYY), which are central to the regulation of glucose metabolism and appetite.\u003c/p\u003e \u003cp\u003eGLP-1 has received considerable attention due to its diverse metabolic effects. It enhances insulin secretion, suppresses glucagon release, slows gastric emptying, and increases satiety, thereby supporting weight loss and improved glycaemic control [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Likewise, GIP and PYY contribute to appetite regulation and energy homeostasis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. By redirecting nutrient flow toward the distal small bowel, bariatric procedures may strengthen these hormonal pathways, increasing the incretin response and supporting metabolic health.\u003c/p\u003e \u003cp\u003eDespite these benefits, a major trade-off remains. Bypassing excessive lengths of the small bowel can result in macronutrient and micronutrient deficiencies due to reduced absorptive length and surface area [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This creates an ongoing clinical challenge for surgeons: optimizing weight loss and metabolic improvement while minimizing malnutrition risk. At present, selecting an appropriate bypass length often depends largely on surgeon experience and retrospective clinical evidence, which remains limited in scientific robustness. Moreover, the methods used to measure bypass length are relatively basic. Distances are commonly estimated using tape or ruler measurements, or laparoscopic instruments, from fixed anatomical landmarks such as the duodenojejunal (DJ) flexure or ileocecal valve, using stepwise hand-over-hand techniques (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, this approach is vulnerable to variability because adult small bowel length differs substantially between individuals. As a result, it remains difficult to develop universally applicable guidelines for bypass length.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese limitations highlight the need for precision medicine in bariatric surgery. By incorporating individual anatomical and physiological characteristics, surgical strategies may be tailored to maximize metabolic benefit while reducing the likelihood of nutritional deficiencies. Progress in understanding the role of L-cells in incretin hormone secretion, together with advances in imaging, could enable more individualized surgical interventions. Ultimately, achieving the optimal balance in bypass length may improve patient outcomes and reduce long-term complications. The primary objective of this study is to evaluate the efficacy of conjugated fluorophore-labelled antibodies for GLP-1, GIP, and SGLT-1 in targeted tissue in vitro, as a prerequisite to subsequent in vivo studies.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThere was clear evidence of absolute uptake of fluorescein\u0026ndash;anti GLP‑1 in the terminal ileum and of IR800CW\u0026ndash;anti GIP in the proximal small bowel (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In both cases, the fluorescence signal was localized to enterocytes as well as to capillaries within the lamina propria and the subserosal layer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFluorescence imaging was performed using an inverted microscope (DMI6000B; Leica Biosystems) equipped with an NIR illumination source (X‑Cite 200DC; Excelitas Technologies) and an NIR filter set comprising band‑pass filters (850\u0026ndash;890 nm) and a long‑pass emission filter (HQ800795LP; Chroma Technology Corp.). Images were acquired with a monochrome DFC365 FX camera (one 4‑million‑pixel charge‑coupled device; Leica Biosystems GmbH) and analysed using LAS‑X software (Leica Biosystems GmbH). For the fluorescein‑conjugated antibody, excitation/emission (Ex/Em) maxima were observed at 495/515 nm at a dilution of 1:200. For the IR800CW‑conjugated antibody, Ex/Em maxima were observed at 773/792 nm when used at a dilution of 1:400.\u003c/p\u003e \u003cp\u003eIn contrast, we did not detect uptake of MB633\u0026ndash;anti SGLT‑1 in any segment of the murine small intestine, even after testing multiple antibody dilutions (1:100, 1:200, and 1:400), with an Ex/Em profile of 630/650 nm. SGLT‑1 is an essential transporter involved in intestinal glucose absorption, primarily localized to the brush border of enterocytes in the duodenum and jejunum. In the intestine, SGLT‑1 mediates glucose transport from the intestinal lumen into epithelial cells, using the sodium gradient generated by the Na⁺/K⁺ ATPase pump [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This glucose transport mechanism is important for efficient nutrient uptake following meals. The most likely explanation for the poor uptake of MB633\u0026ndash;anti SGLT‑1 is inefficient antibody binding to SGLT‑1 in the small intestine, which may reflect low or altered SGLT‑1 expression and/or reduced epitope accessibility on the tissue surface. Additionally, the MB633 conjugation may impair antibody binding through steric hindrance or an unfavourable epitope\u0026ndash;dye orientation. Other contributing factors may include species/isoform mismatch or luminal interference (e.g., mucus or digestive components) that could further limit effective antibody binding.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSmall-bowel physiological changes after bariatric surgery reflects that the small intestine is both an absorptive organ and an enteroendocrine signalling system, with function determined by where nutrients are delivered and when they encounter digestive secretions and hormone-secreting cells. Bariatric procedures reconfigure nutrient transit, the timing of mixing between food, bile and pancreatic enzymes, and the luminal environment that shapes both absorption and gut-hormone responses. Ngo et al. reported that anatomical variation in small-bowel length is significantly associated with glycaemic control and metabolic status [9], helping explain why outcomes differ between patients even after the same operation. In RYGB, bypass of the duodenum and proximal jejunum reduces proximal nutrient exposure while accelerating distal delivery of carbohydrates and fats, which enhances distal enteroendocrine stimulation and GLP‑1 mediated effects that improve appetite regulation and glucose homeostasis; delayed mixing can also transiently impair fat digestion and micelle formation. In DS/BPD‑DS, these effects are amplified by greater separation of nutrient flow from bile/pancreatic enzymes and a shorter common channel, increasing the likelihood of clinically meaningful malabsorption, especially fats and fat-soluble vitamins, and in severe cases protein. Sleeve-plus procedures are typically less malabsorptive than DS/BPD‑DS, but altered gastric emptying and bile acid–nutrient dynamics can still shift incretin profiles and promote micronutrient deficiencies depending on the specific “plus” anatomy [10].\u003c/p\u003e\n\u003cp\u003eMajor pitfalls arise when intake and supplementation do not match the post-surgical absorptive capacity. Severe protein deficiency may result from inadequate protein intake (low dietary protein, supplement intolerance, vomiting, poor adherence) and from reduced effective absorption due to anatomy or complications such as bacterial overgrowth. In DS, the shortened absorptive window increases vulnerability to hypoalbuminemia, sarcopenia, and systemic consequences of protein-calorie malnutrition, particularly when chronic diarrhoea, surgical stress, or SIBO further impair nutrient handling. Micronutrient deficiencies follow predictable mechanistic patterns: iron and B-vitamins can decline with reduced exposure to proximal absorptive regions and altered digestion/acid conditions; calcium and vitamin D may deteriorate when absorption is less efficient; and fat-soluble vitamins (A, D, E, K) are especially at risk when fat digestion/absorption is impaired by mixing delay or common-channel shortening [11]. Across bypass configurations, SIBO can worsen both macronutrient and micronutrient depletion by promoting luminal overgrowth and impaired digestion.\u003c/p\u003e\n\u003cp\u003eVariation in outcomes is therefore driven not only by procedure type, but also by the interaction of anatomy, small-bowel morphology, microbiome, and adaptive capacity over time. Limb lengths and surgical technique determine common-channel length and the site where nutrients meet bile and pancreatic enzymes, while adherence and tolerance determine nutrient delivery. Microbiome shifts and bile acid changes further modulate nutrient processing, barrier function, and hormone signalling, and complications such as SIBO, strictures, or chronic diarrhoea can alter effective absorption and symptom profiles even after technically successful surgery.\u003c/p\u003e\n\u003cp\u003ePrecision medicine can be strengthened by mapping the location and density of SGLT‑1, GLP‑1 secreting cells (L cells), and GIP-secreting cells (K cells). Because SGLT‑1 mediates enterocyte glucose uptake, segment-specific glucose delivery after surgery may help predict absorption efficiency, glycaemic patterns, and dietary tolerance. Distal nutrient delivery generally enhances GLP‑1 signalling, supporting satiety and improved insulin secretion, whereas K cells are more prominent proximally; thus, foregut exclusion in RYGB can reduce proximal K-cell nutrient exposure and shift incretin balance. This spatial framework can support prevention by anticipating which nutrients will contact impaired absorptive/endocrine regions, tailoring protein/carbohydrate strategies, intensifying early monitoring of high-risk micronutrients, and evaluating/treating SIBO when luminal clearance is likely impaired.\u003c/p\u003e\n\u003cp\u003eIn summary, identifying the distribution and density of GLP‑1, GIP, and SGLT‑1 could guide bypass length selection in RYGB, DS/BPD‑DS, and sleeve-plus to optimize metabolic benefits while minimizing malabsorption. Moving forward, we plan in vivo real-time fluorescent imaging with Cellvizio to map the intra-operative distribution and density of GLP‑1, GIP, and SGLT‑1 in rats’ small intestine and use this information to refine bypass design to prevent nutrient malabsorption [12].\u003c/p\u003e"},{"header":" Methods","content":"\u003cp\u003e\u003cstrong\u003e1. Animal Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale 12-week-old Wistar rats were housed in a barrier facility on high-efficiency particulate air (HEPA)-filtered racks in a controlled room maintained at 22 °C under a 12-h light/dark cycle (lights on at 07:00 and off at 19:00). Animals were fed autoclaved laboratory rodent diet (Teckland LM-485; Western Research Products, Laramie, WY). In this in vivo study, rats were humanely sacrificed for the collection of small intestine tissue samples. As the experimental procedure did not involve any surgical intervention or invasive manipulation prior to tissue harvesting, anaesthesia was not required. Animals were euthanized using cervical dislocation to ensure rapid and humane death. Immediately after euthanasia, the abdominal cavity was opened and the small intestine was carefully removed, rinsed in cold phosphate-buffered saline (PBS) to remove residual luminal contents, and segmented for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Ethical Considerations and Animal Welfare\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols were reviewed and approved by the SingHealth Institutional Animal Care and Use Committee (IACUC), and all procedures were conducted in accordance with institutional and national ethical approvals. Prior to study initiation, the experimental design was reviewed to confirm scientific necessity and the absence of viable alternatives. Throughout the study, animal welfare was prioritized by minimizing discomfort, providing appropriate housing and nutrition, and ensuring veterinary monitoring. Personnel were trained in humane handling and animal care to support ethical conduct while enabling scientific objectives.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Antibodies Conjugation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMonoclonal antibodies specific for GLP-1, GIP, and SGLT-1 were obtained from Thermo Fisher Scientific (Waltham, MA). Antibodies were conjugated with either the IRDye 800CW or AlexaFluor 488 Protein Labelling Kit (Molecular Probes, Grand Island, NY) according to the manufacturer’s instructions [13]. Briefly, antibodies were reconstituted at 1 mg/mL in 0.1 M sodium bicarbonate. One hundred microliters of the antibody solution were added to the reactive dye and incubated for 1 hour at room temperature. Conjugated antibodies were then separated from unreacted dye using a gravity purification column. Antibody and dye concentrations in the final preparations were determined by spectrophotometric absorbance measurements (\u003cstrong\u003eFigure 4\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Immunofluorescence Staining of Formalin-Fixed Paraffin-Embedded Tissue Sections\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn vitro testing using target tissue sections involves preparing different segments of the small intestine- proximal, mid, and distal that express GLP-1, GIP, and SGLT-1. Slides were deparaffinised through sequential xylene and ethanol washes using Coplin jars. Sections were incubated in xylene for 9 minutes and then in fresh xylene for an additional 9 minutes. Dehydration was performed using a graded ethanol series: 100% ethanol for 5 minutes (twice), 95% ethanol for 5 minutes (twice), 85% ethanol for 5 minutes (twice), and 75% ethanol for 5 minutes (twice). Following dehydration, slides were washed three times in water (5 minutes per wash). After the final water wash, slides were kept hydrated until antigen retrieval was performed; drying at any point was avoided because it increased non-specific antibody binding and elevated background staining.\u003c/p\u003e\n\u003cp\u003eFor antigen retrieval, tissue sections were encircled with a PAP pen to localize reagents to the section. A working solution of proteinase K was prepared by diluting proteinase K in TE buffer (pH 8.0) containing 0.5% Triton-X at a 1000× dilution. Three hundred microliters of the working solution were applied to each section and incubated for 15 minutes at 37 °C. Slides were then washed with TBST (400 µL per wash) three times for 5 minutes each. For antibody incubation, slides were blocked with 400 µL of blocking buffer (1× TBST + 2% BSA) for 1–2 hours. Blocking buffer was removed, and fluorescence-conjugated primary antibodies were applied at the indicated dilutions (Fluorescein–anti GLP1 1:200; IR800CW–anti GIP 1:400; MB633–anti SGLT-1 tested at 1:100, 1:200, or 1:400). Sections were incubated with 300 µL of primary antibodies overnight at 4 °C. Primary antibodies were then removed and slides were washed three times with TBST (400 µL per wash) for 5 minutes each. After the final wash, excess TBST was removed by gently dabbing the slides dry, and antifade mounting media (2-3 drops) was applied prior to placing glass coverslips (\u003cstrong\u003eFigure 5\u003c/strong\u003e).\u0026nbsp;Fluorescence imaging is utilized to visualize the binding of the conjugated antibodies, capturing images to assess localization and intensity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study involving animal subjects has been conducted in accordance with ethical guidelines. Written informed consent and approval from the Institutional Review Board (IRB) was deemed unnecessary given the nature of the study. All procedures were performed in compliance with relevant regulations to ensure the welfare of the animals involved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw data and analysis code are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLCH, BY and LJL conceived the experiments. LCH, CL, CKM, TNG and LO conducted the experiments. LCH, CWH, RA and JT analysed the results. LCH wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) received no financial support for the research, authorship, and/or publication of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSjostrom, L. et al. Effects of Bariatric Surgery on Mortality in Swedish Obese Subjects. \u003cem\u003eNEJM\u003c/em\u003e \u003cb\u003e357\u003c/b\u003e, 741\u0026ndash;752 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchauer, P. et al. Bariatric surgery versus intensive medical therapy for diabetes- 5-year outcomes. \u003cem\u003eN Eng. J. 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Associations of Bariatric Interventions With Micronutrient and Endocrine Disturbances. \u003cem\u003eJAMA Open.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e (6), e205123 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNgo, C. et al. Association of Total Small Bowel Length with Glycaemic Control and Metabolic Syndrome in Metabolic and Bariatric Surgery Patients: A Cross-Sectional Study in a Taiwanese Cohort. \u003cem\u003eObes. Surg.\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11695-026-08594-5\u003c/span\u003e\u003cspan address=\"10.1007/s11695-026-08594-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2026).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurahashi, Y., Hojo, Y. \u0026amp; Shinohara, H. Sleeve Plus Procedures: A Review of Technical Evolution Along a Schematic Diagram. \u003cem\u003eObes. Surg.\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e, 4806\u0026ndash;4813 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLewis, C. A. et al. Monitoring for micronutrient deficiency after bariatric surgery\u0026mdash;what is the risk? \u003cem\u003eEur. J. Clin. Nutr.\u003c/em\u003e \u003cb\u003e77\u003c/b\u003e, 1071\u0026ndash;1083 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePohl, H. et al. Miniprobe confocal laser microscopy for the detection of invisible neoplasia in patients with Barrett's oesophagus. \u003cem\u003eGut\u003c/em\u003e \u003cb\u003e57\u003c/b\u003e, 1648\u0026ndash;1653 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, M., Li, N. \u0026amp; Zhou, H. SGLT1: A Potential Drug Target for Cardiovascular Disease. \u003cem\u003eDrug Des. Devel Ther.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 2011\u0026ndash;2023 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bariatric surgery, GLP-1, GIP, SGLT-1, Immunofluorescence, Fluorescent antibodies","lastPublishedDoi":"10.21203/rs.3.rs-9251347/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9251347/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBariatric surgery provides substantial weight loss and metabolic improvements in severe obesity. RYGB, DS, OAGB, and SADI-S share a mechanism of diverting nutrient flow away from the duodenum and proximal jejunum, altering gut hormone signalling and incretin responses. However, bypass length is guided largely by surgeon experience and retrospective evidence, limiting precision due to variability in individual small-bowel anatomy. Target-specific visualization of enteroendocrine and absorptive markers could enable more precise bypass design. We evaluated whether conjugated fluorophore-labelled antibodies against GLP-1, GIP, and SGLT-1 can be detected in vitro in rat small-intestinal tissue as a prerequisite for subsequent in vivo studies. Male 12-week-old Wistar rats were euthanized for intestinal segment collection; monoclonal antibodies were conjugated with IRDye 800CW or Alexa Fluor 488 and applied to formalin-fixed, paraffin-embedded small-bowel sections using immunofluorescence. Fluorescein\u0026ndash;anti GLP-1 showed uptake localized to the terminal ileum, while IR800CW\u0026ndash;anti GIP localized to the proximal small bowel, with signal observed in enterocytes and in lamina propria vasculature and subserosal layers. In contrast, MB633\u0026ndash;anti SGLT-1 showed no detectable uptake across tested segments or dilutions (1:100\u0026ndash;1:400), potentially due to limited epitope accessibility, altered SGLT-1 expression, steric effects, and/or species or isoform mismatch. Conjugated antibodies enabled segment-specific in vitro detection of GLP-1 and GIP but not SGLT-1. These results support the feasibility of fluorophore-labelled incretin-marker imaging (Cellvizio or fluorescence laparoscopy) for future intra-operative mapping, with the long-term goal of refining personalized bypass design while minimizing malnutrition risk.\u003c/p\u003e","manuscriptTitle":"Fluorophore-Conjugated Antibodies Enable In Vitro Mapping of GLP-1 and GIP in Rat Small Intestine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 10:54:37","doi":"10.21203/rs.3.rs-9251347/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-05-01T14:36:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-01T14:30:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-29T14:24:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-11T10:27:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-11T10:23:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ec09191c-b5e9-4c96-84aa-afc3144da1b2","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"4","date":"2026-05-01T14:36:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-01T14:30:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-29T14:24:58+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67445212,"name":"Health sciences/Diseases"},{"id":67445213,"name":"Health sciences/Gastroenterology"},{"id":67445214,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-05-11T10:54:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 10:54:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9251347","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9251347","identity":"rs-9251347","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}