Pgc 1α

Immunohistochemistry was conducted on 4 μm paraffin-embedded renal tissues. Antigens were extracted by boiling in citrate buffer after deparaffinization and rehydration. Sections were blocked for 15 min with 0.3% H₂O₂ and 1 h with 5% BSA. Primary antibodies of Drp1 (1:200, Abcam, United States), Mfn2 (1:250, CST, USA), Fis1 (1:200, Abcam, United States), Sirt1 (1: 200, Abcam, USA), PGC-1α (1:200, ABclonal, China) were incubated overnight at 4°C. Then the sections were incubated with the secondary antibodies (Dako, United States) for 1 h at 37°C. Finally, the sections were counterstained with diaminobenzidine and hematoxylin. A light microscope (Leica, Germany) was then used to collect photomicrographs. And all photomicrographs were analyzed by ImageJ software.

RIPA lysis buffer (Beyotime Tech, China) containing phosphatase and protease inhibitors was used to extract proteins from kidney tissues and cells. The tissue lysates were mixed with 5 × SDS buffer and denatured at 100 °C for 10 min. The samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-page) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% nonfat milk in TBST for 1 h at room temperature. Primary antibodies of Bcl-2 (1:1,000, Abcam, United States), Bax (1:1,000, CST, United States), cleaved-caspase-3 (1:500, CST, United States), Drp1 (1:1,000, Abcam, United States), Mfn2 (1:1,000, CST, United States), Fis1 (1:500, Abcam, United States), Sirt1 (1:1,000, Abcam, United States), PGC-1α (1:1,000, ABclonal, China), β-actin (1:5,000, Abcam, United States) were incubated with membranes overnight at 4°C. After washing with TBST for 5–10 min, the membranes were incubated at room temperature for 1 h with HRP combined secondary antibody (Abcam, United States) and then washed with TBST for 10 min for 3 times. Finally, the proteins bands were visualized with ECL and the band intensity was measured with ImageJ software.