Quantifying Cardiomyocyte Apoptosis by TUNEL

Terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end labeling (TUNEL) staining was conducted to detect apoptotic nuclei by confocal microscopy in α-actinin-labeled cardiomyocytes as described before [21 (link)]. Briefly, neonatal rat cardiomyocytes (NRCMs) or frozen mouse heart sections were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100 in PBS, and blocked with 5% bovine serum album (BSA) before incubation with mouse anti-α-actinin (Sigma, A7811, 1:200 dilution). After incubated with Cy3-AffiniPure goat anti-mouse IgG (H+L) (Jackson), cells or tissue sections were stained with TUNEL FITC Apoptosis Detection Kit (Vazyme) according to the manufacturer’s instructions. Nuclei were counterstained with DAPI. Finally, 20–30 fields per sample were viewed under a confocal microscope (Carl Zeiss). The percentage of TUNEL-positive cardiomyocytes was calculated to determine apoptosis induced by OGDR or I/R injury.