Immunohistochemical assay

The paraffin-embedded kidney sections were deparaffinized, subjected to antigen retrieval and blocked. Antigen retrieval was performed in high-pressure citrate buffer (0.01 mol/L, pH 6.0) for 10 min, and the retrieved antigen was blocked with 10% goat serum for 30 min at room temperature. After incubation with 3% hydrogen peroxide for 10 min, the sections were incubated with anti-Sirt6 antibody (1:100, Thermo Fisher Scientific, USA) overnight at 4 °C. Then, the sections were incubated with polymerized horseradish peroxidase-conjugated secondary antibody for 30 min. After diaminobenzidine staining, the sections were counterstained with hematoxylin. Slides were viewed by microscopy (Olympus, Japan).