Immunofluorescent staining

For fluorescent microscopy imaging of insulin, cleaved-caspase 3, and Ki67, paraffin-embedded tissue sections were deparaffinized in xylene, hydrated through graded ethanol to distilled water, and permeabilized in PBS with 0.1% Triton X-100 (PBST) for 10 min. Blocking was performed using 5% bovine albumin serum (BSA) in PBST for 1 h at RT, followed by incubation with guinea pig polyclonal anti-insulin antibody (Abcam, Cambridge, UK) together with rabbit anti-cleaved caspase-3 (Cell Signaling Technology) or rabbit anti-Ki67 (Abcam) diluted 1:100 in 1% BSA in PBST at 4°C overnight. Three consecutive washes with PBST for 5 min each were followed by sequential incubation with either Alexa Fluor 488 goat anti-guinea pig or Alexa Fluor 594 goat anti-rabbit secondary antibodies (Invitrogen) diluted 1:300 in 1% BSA in PBST at RT for 1 h. The slides were washed 3 times with PBST and mounted using a prolonged diamond anti-fade mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen). Images were captured under a fluorescence microscope (Olympus D73 equipped with CellSens software). Positive staining for insulin in pancreatic sections was quantified at 630-fold magnification and expressed as the percentage of insulin-positive cells divided by islet area multiplied by 100. In addition, quantification of the percentage of islets containing apoptotic β-cells was performed by the manually counted number of cleaved-caspase 3 positive staining cells divided by the total number of DAPI stained cells multiplied by 100. Lastly, the percentage of cell proliferation was determined by the number of coexpressing insulin and Ki67 cells divided by the insulin fluorescent intensity within each islet core. National Institutes of Health (NIH) ImageJ software was employed to measure fluorescent intensity. At least 5 different islets per pancreas section and 3 to 4 mice per condition were counted.