G-protein coupled short-chain fatty acid receptor GPR 43 fluorescence immunohistochemistry staining protocol for confocal microscopy in human colon cancer

Background G-protein coupled receptor 43 (GPR43) mediates the biological effects of short-chain fatty acids (SCFAs) in the colon, but its role in colon cancer (CC) remains insufficiently characterized. This pilot study presents in detail a cost-effective and easily reproducible fluorescence immunohistochemistry protocol for staining GPR43 in formaldehyde-fixed, paraffin-embedded human colon tissue. Methods Using samples from patients with colon adenocarcinoma (ACC) and signet ring cell carcinoma (SRCC), we optimized staining procedures with generic reagents and a custom 3D-printed slide tray, facilitating minimal reagent usage and consistent sample coverage. Results Confocal microscopy revealed distinct GPR43 localization on the apical and basal membranes of colonocytes and goblet cells in healthy tissue. In contrast, ACC tissue exhibited diffuse, disorganized staining, while SRCC samples showed minimal receptor expression. These findings suggest a link between GPR43 expression patterns and tissue differentiation status. Additionally, we observed autofluorescence at 488 nm of eosinophils without staining, as previously reported in literature. Conclusions These findings may support prognostic assessments based on intratumoral or peritumoral eosinophil counts. The present study shows in detail an easily reproducible IHC staining method for GPR 43 in paraffin embedded tissue samples, using only generic materials and highlights its potential role as a biomarker or therapeutic target in CC.