Anti sirt3 antibody

Resveratrol was purchased from Aladdin (Shanghai, China). NAC (N-acetyl-L-cysteine) and Dimethyl sulfoxid (DMSO) were purchased from Sigma (St. Louis, MO, USA). The MDA assay kit, SOD activity assay kit, antioxidant capacity assay kit, glutathione peroxidase assay kit and glutathione reductase assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nangjing, China). Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (St Louis, MO, USA). A cell counting kit (CCK-8) was purchased from Biosharp (Hefei, China). Annexin V-FITC/PI staining kit was purchased from BestBio (Shanghai, China). Anti-MnSOD antibody, anti-Sirt3 antibody, anti-LC3A/B antibody and anti-Beclin1 antibody were purchased from Abcam (Beverly, MA, USA). Alexa Fluor 488-conjugated secondary antibody and 4′,6′-diamidino-2-phenylindole (DPI) were purchased from BEIJING BIOSS (beijing, China). MitoSOX Red mitochondrial superoxide indicator and 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) for live-cell imaging was obtained from life technologies (San Diego, CA, USA).

FLSs were grown on glass coverslips and then harvested. The coverslips were fixed with 4% (v/v) paraformaldehyde for 30 min at 37 °C and permeabilized with 0.1%Triton X-100 for 20 min at room temperature. The slides were blocked with Immunol Staining Blocking Buffer (Beyotime, Shanghai, China), incubated with anti-LC3A/B antibody, anti-MnSOD antibody, anti-Sirt3 antibody (1:200 dilution, Abcam, MA, USA), anti-Beclin1 antibody (1:300 dilution, Abcam, MA, USA) overnight at 4 °C. After washing with PBS, the slides were incubated for 40 min at 37 °C with a Alexa Fluor 488-conjugated secondary antibody (1:400 dilution; BEIJING BIOSS, Bengjing, China). Finally, the slides were washed twice with PBS and the nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI, BEIJING BIOSS, Bengjing, China) for 4 min at room temperature. Images of the stained cells were obtained using confocal laser scanning microscopy (model LEICA.SP5-DMI6000-DIC; Leica Microsystems GmbH) and the fluorescence intensity was expressed as the percentage relative to the control group (set as 100%), respectively.

Immunohistochemical staining was performed as described [43 (link)]. Paraffin-embedded kidney tissue sections were stained with anti-SIRT3 antibody (1:100, Abcam, Cambridge, UK) at 4 °C overnight. Graphs were obtained by using a Ti2-A fluorescence microscope (NIKON, Tokyo, Japan).