Immunofluorescence Staining of Pancreatic Insulin and Glucagon

Paraffin embedded tissues were sectioned at 5–6 μm and OCT tissues were cryosectioned (Thermoscientific, UK) at 8–10 μm thickness between -14 to -30°C. All tissues were haematoxylin and eosin (H&E) stained, gut and liver tissue sections were also stained with Gömöri’s Trichrome using previously described methods [12 (link)]. Prior to immunofluorescence (IF) staining, antigen retrieval (10 mM citrate buffer, pH 6) was performed and the sections were avidin/biotin blocked (Vectorlabs, UK). Briefly, pancreatic insulin and glucagon were stained with rabbit anti-insulin (1/200 dilution: Catalogue number Ab181547 Abcam, UK) and mouse anti-glucagon antibodies (1/200 dilution: Catalogue number Ab10988 Abcam, UK) overnight, followed by goat anti-rabbit IgG Alexafluor 488 (1/400 dilution: Abcam, UK) and goat anti-mouse IgG Alexafluor 647 (1/400 dilution: Catalogue A28181 Invitrogen, UK). IL-17 was detected using goat anti-mouse IL-17 (1/100 dilution: Catalogue number Af-421-na R&D Systems, UK) followed by biotinylated rabbit anti-goat IgG (1/200 dilution: Catalogue number 31732 Invitrogen, UK) with streptavidin Alexafluor 647 (1/200: dilution Catalogue number S21374 Invitrogen, UK). IF sections were mounted with Vectashield with DAPI (Vectorlabs, UK) and images were acquired with an EVOS FL Auto 2 system (Thermofisher, UK).