Live cell observer microscope

Kit^MerCreMer/R26^YFP fate-mapping mouse was transcardially perfused with 15 ml 4% paraformaldehyde. The pancreas was excised, immersed in 4% paraformaldehyde overnight, followed by dehydration in 30% sucrose before being embedded in OCT. Eight μm sections were cut and stained with anti-Iba1 antibody followed by donkey anti-rabbit Alexa Fluor647. Images were taken by a Zeiss Live Cell Observer microscope.

The pancreas was fixed in 10% formalin at autopsy followed by dehydration in 30% sucrose and then embedded in Optimal Cutting Temperature (O.C.T.) compound (Tissue Tek, Sakura Finetek, Torrance, CA, USA) for subsequent analysis. ten μm sections were cut and stained with anti-mouse insulin antibody (Cell Signaling Technology Inc., Danvers, MA, USA) followed by Donkey anti-rabbit Alexa Fluor647 (Biolegend) and analysed by a Zeiss Live Cell Observer microscope. The islet size was examined by compiling measurements of 30 insulin-stained pancreas islets using ImageJ and Zeiss Zen Blue software. Islet macrophages were stained with rabbit anti-Iba1 antibody (dilution 1:400, FUJIFILM Wako Chemicals, Richmond, VA, USA) followed by Donkey anti-rabbit Alexa Fluor647 (dilution 1:300, Biolegend). Their numbers were manually counted from 18-21 islet images taken with the microscope. Three animals for each group were analysed.