Pancreases were dissected, weighed, fixed in 4% formalin overnight at 4 °C, washed with phosphate buffered saline (PBS), and embedded in paraffin. The pancreatic sections were stained as previously described 23 (link) using SIRT2 (Abcam, ab67299), insulin (Abcam, ab7842), and glucagon (Abcam, ab10988) antibodies. For α/β cell number statistics, 7-10 5 μm sections of pancreas (paraffin embedded, n=3 per genotype), separated by at least 200 μm, were co-stained for insulin and glucagon. The number of α/β cells per islet on the section level was determined by counting the glucagon+ /insulin+ cells manually.
INS-1 cells were fixed for 20 min in 4% formalin, permeabilized in 0.1% Triton×100 for 5 min, washed with PBS, and blocked in 5% BSA for 1 h. Cells were then incubated with anti-GKRP antibody (1: 100) overnight at 4 °C and stained with FITC-labeled IgG (1: 200, Jackson ImmunoResearch Laboratories). DAPI was added to stain cell nuclei. The cellular localization of GKRP was photographed and analyzed using a fluorescence microscope (LSM 880 with Airyscan, ZEISS).
INS-1 cells were fixed for 20 min in 4% formalin, permeabilized in 0.1% Triton×100 for 5 min, washed with PBS, and blocked in 5% BSA for 1 h. Cells were then incubated with anti-GKRP antibody (1: 100) overnight at 4 °C and stained with FITC-labeled IgG (1: 200, Jackson ImmunoResearch Laboratories). DAPI was added to stain cell nuclei. The cellular localization of GKRP was photographed and analyzed using a fluorescence microscope (LSM 880 with Airyscan, ZEISS).