Dab kit

After 4-µm kidney sections were deparaffinized and hydrated, each section was exposed to 3% hydrogen peroxide solution. After antigen repair, kidney sections were incubated with primary antibodies against γ H2AX (1:200, Abcam, ab2893) overnight at 4°C. Subsequently, sections were incubated with HRP-conjugated secondary antibody (Proteintech Group, Inc., Chicago, IL, United States) at room temperature for 30 min; a DAB kit (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) was used for localization. Images were taken through a microscope (×400).

For immunohistochemistry staining, kidney tissues were fixed, deparaffinized, blocked, and reduced nonspecific binding by 0.1% Triton X-100. The slides were then incubated with primary antibodies at 4°C overnight and counteracted with HRP-conjugated secondary antibody for 1 hour at room temperature. The color was developed with a DAB kit (Beijing Zhongshan Jinqiao Biotechnology, ZLI-9018).

Paraffin-embedded kidney tissues were sectioned at a thickness of 4 μm for IHC analysis. After dewaxing, rehydration, blocking, and antigen retrieval, the kidney tissue sections were exposed to anti-apelin antibodies (1:100) at 4 °C overnight. Then the sections were incubated with biotinylated goat anti-rabbit secondary antibodies (PV-9000, Zhongshan Jinqiao Biotechnology, Beijing, China) for 30 min at room temperature. A DAB kit (ZLI-9018, Zhongshan Jinqiao Biotechnology, Beijing, China) was used to detect the signal of the antigen-antibody complexes. Finally, the slides were counterstained in hematoxylin.