Paraffinized sections were deparaffinized and rehydrated, and antigen was retrieved with 10 mM Tris/EDTA (pH 9.0). The sections were permeabilized and blocked in PBS containing 1% BSA and 5% goat serum. Primary antibodies were incubated overnight at 4°C, followed by secondary antibodies incubation at 37°C for 1 hour. For immunofluorescence staining, anti-insulin antibody (Abcam, Cambridge, UK) and anti-glucagon antibody (Abcam) were used to detect β and α cells, respectively. Pancreatic sections were stained with anti-insulin antibody (HUABIO, Hangzhou, China) and anti-PCNA antibody (HUABIO), followed by tyramine amplification with fluorophores 488 and 594 and DAPI counterstaining to determine the proliferation of β cells. Insulin-positive (insulin+) cells showing nuclear colocalized staining for DAPI+ and PCNA+ were considered as proliferating β cells. Sections were visualized using a confocal microscope (LSM 880; Carl Zeiss, Oberkochen, Germany).
For immunohistochemistry staining, pancreatic sections were incubated with anti-insulin antibody (Abcam), followed by binding with horseradish peroxidase (HRP)-conjugated secondary antibodies, and detected with a DAB Kit and counterstained with hematoxylin. Quantification was performed using ImageJ.
For immunohistochemistry staining, pancreatic sections were incubated with anti-insulin antibody (Abcam), followed by binding with horseradish peroxidase (HRP)-conjugated secondary antibodies, and detected with a DAB Kit and counterstained with hematoxylin. Quantification was performed using ImageJ.