Insulin alexa fluor 488

Tissues were fixed (10% formalin solution in 0.1 M PBS), paraffin embedded, and cut into 4-μm sections on a microtome. Tissue sections were stained with hematoxylin and eosin (H&E) under standard conditions. MIN6 cells were fixed in 2% paraformaldehyde for 40 min. Both the sections and cells were permeabilized in 0.1% Triton X-100 in PBS and blocked in 5% normal goat serum and 0.1% Triton X-100 in PBS prior to incubation with primary antibody. Islets were stained using Sirt6 Ab (Cell Signaling Technology, Beverly, MA, USA), Glut2 Ab (Santa Cruz Biotechnology, Dallas, TX, USA), glucagon Ab (Bioworld Technology, St Louis Park, MN, USA), and insulin Alexa Fluor® 488 (eBioscience, San Diego, CA, USA) together with DAPI nuclear stain (Invitrogen). Images were acquired using an Axiovert 40 CFL confocal microscope (Carl Zeiss, Oberkochen, Germany). Islet size was measured using iSolution DT 36 software (Carl Zeiss).