3 3 diaminobenzidine dab staining

Immunohistochemistry staining was performed as previously described56 (link). The cardiac tissue was fixed with 10% formalin and embedded in paraffin. Anti-PGC-1α antibody (Cell Signaling Technology, MA, USA, 1:100 dilution), anti-SIRT3 antibody and anti-SOD2 antibody (Santa Cruz, CA, USA, 1:100 dilution) were used as the primary antibody. The positive area was detected by 3,3′-Diaminobenzidine (DAB) staining (Zhongshan biotechnology, Beijing, China). IgG antibody was used as a negative control. The images were photographed at 200× magnification (Olympus BX-63, JAPAN).

The nitrotyrosine content was also measured by immunohistochemistry staining. The detailed protocol was described in our previous publications [21 (link), 22 (link)]. The LV tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Then, they were cut into 3 mm thickness and stained with primary antibody (anti-nitrotyrosine antibody, Cell Signaling Technology, MA, USA, 1 : 200 dilution). Then, the sections were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies (Zhongshan Biotechnology, Beijing, China) and detected with 3,3′-diaminobenzidine (DAB) staining (Zhongshan Biotechnology, Beijing, China). Five fields of each section were randomly chosen and photographed at ×200 magnification (Olympus BX-63, Tokyo, Japan). The graphs were analyzed and calculated using Image-Pro Plus software (Media Cybernetics, MA, USA).