Citrate buffer

The tissue sections were deparaffinized in xylene (Sinopharm Chemical Reagent Co., Ltd.), hydrated with 100% and 95% ethanol (Sinopharm Chemical Reagent Co., Ltd.), and then rinsed in distilled water. Endogenous peroxidase was blocked with 0.1% H₂O₂ (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 20 min. The sections were prepared by microwave antigen retrieval in 10 mM citrate buffer (pH 6.0; Zhongshan Golden Bridge Biotechnology Co., Ltd.) for 10 min. The slides were subsequently incubated with serum blocking solution (Zhongshan Golden Bridge Biotechnology Co., Ltd.) for 1 h at 37°C, rabbit anti-human polyclonal anti-Wip1 primary antibody (catalog no., SC-20712; 1:100 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C, goat anti-rabbit biotinylated secondary antibody (catalog no., ZB2010; dilution, 1:100; Zhongshan Golden Bridge Biotechnology Co., Ltd.) for 1 h at 37°C and streptavidin-horseradish peroxidase. 3,3′-Diaminobenzidine solution (Sigma-Aldrich) was used as a chromogen. The slides were then counterstained in a hematoxylin solution (Sigma-Aldrich) and visualized on the DM IL LED microscope (Leica Microsystems GmbH). Negative controls were performed by omitting the primary antibody incubation step.

HE staining was applied to evaluate the morphological changes in pancreatic microcirculation. After fixing with 4% paraformaldehyde, pancreatic tissues were embedded in paraffin and cut into five‐micrometer‐thick sections for HE staining and immunohistochemical staining. Briefly, sections were deparaffinized, rehydrated, and incubated in boiling 10 mM citrate buffer (pH = 6.0, Zhongshan Golden Bridge Biotechnology, Beijing, China) for antigen retrieval. And then, sections were incubated with 3% hydrogen peroxide to inhibit endogenous peroxidase, followed by blocking with 3% bovine serum albumin (BSA, TBD Science Technology, Tianjin, China) in phosphate‐buffered saline. After incubating with primary mouse monoclonal antibody raised against insulin (2D11‐H5) (dilution 1: 50; Santa Cruz Biotechnology, Dallas, TX) and PECAM‐1 (dilution 1: 50; Santa Cruz Biotechnology), sections were incubated in blocking buffer overnight at 4°C. Then, horseradish peroxidase–conjugated secondary antibody (Zhongshan Golden Bridge) and 3,3'‐diaminobenzidine tetrahydrochloride solution (Zhongshan Golden Bridge) were employed for the incubation of sections, which was monitored on a microscope. Finally, the sections were dehydrated and mounted. Positive staining of insulin and PECAM‐1 were determined using a Leica DFC450 microscope (Leica Microsystems, Leitz, Germany).