Dab dab 1031

After deparaffinization and hydration, tissue sections (5 μm) were treated in antigen retrieval solution. Sections were then treated with 3% H₂O₂ (10011218, Sinopharm Chemical Reagent Co., Ltd., China) for 15 min to block endogenous peroxidase activity, and then incubated with 1% BSA (A602440-0050, Sangon Biotech, China) to block nonspecific antibody binding. Primary antibody was added to the sections at 4 °C overnight, followed by HRP-labeled goat anti-rabbit IgG (1:500, #31460, ThermoFisher, USA) at 37 °C for 1 h. Sections were then treated with DAB (DAB-1031, Maixin-Bio, China) and hematoxylin (H8070, Solarbio, China). Images were taken under a microscope (BX53, OLYMPUS, Japan). Antibody information: NREP (1:100, DF14359, Affinity, China), Ki67 (1:100, AF0198, Affinity, China), CD31 (1:100, A4900, Abclonal, China).

For PCNA staining, the slides were incubated at 65°C for 2 h and dewaxed with xylene (15 min, 15 min), alcohols (100% 5 min, 100% 3 min, 95% 3 min, 80% 3 min), and water (5 min, 5 min). Slides were soaked in a pH 6.0 citrate solution at 95°C for 12 min and naturally cooled for 30 minutes. Next, the slides were incubated with endogenous peroxidase blocker (Kit-7310 reagent 1, Maixin, Fuzhou, China) for 15 min at 37°C, nonspecific staining blocker (Kit-7310 reagent 2, Maixin, Fuzhou, China) for 60 min, anti-PCNA antibody (A0264, ABclonal, Wuhan, China) overnight at 4°C, biotin-labeled goat anti-mouse/rabbit IgG polymer (Kit-7310 reagent 3, Maixin, Fuzhou, China) for 30 min at 37°C, and streptavidin-peroxidase (Kit-7310 reagent 4) for 15 min at 37°C. Diaminobenzidine (DAB, DAB-1031, Maixin, Fuzhou, China) staining is used for the brown color and hematoxylin staining for the background. Seal the slides with neutral gum.