Senescence-Associated β-Galactosidase Staining in Human Cardiomyocytes

For SA-β-Gal staining, hCMs were fixed in 2% formaldehyde and 0.2% glutaraldehyde at RT for 5 min and stained with freshly prepared staining solution at 37°C for 9–10 h (X-gal was purchased from Amresco, all the other reagents were from Sigma-Aldrich) (Bi et al., 2020 (link)). After SA-β-gal staining, cells were permeabilized with PBS containing 0.4% Triton X-100 for 5 min at RT. Subsequently, cells were blocked with 10% donkey serum for 30 min. The cardiomyocytes were incubated with primary antibody (cTnT, Abcam) overnight at 4°C, and then the fluorescent-dye conjugated secondary antibody at RT for 1 h. Images were taken with an Olympus CKX41 microscope, and the percentages of SA-β-gal-positive cells were quantified using Image J.