Terminal deoxynucleotide nick-end labeling (TUNEL) assay

Myocardium apoptosis was assessed with an in situ apoptosis detection kit (S7101, Millipore, USA) through the immunohistochemical method according to the manufacturer's instructions. The percentage of apoptotic cells was determined by calculating the ratio of positive-staining nuclei to the total number of nuclei.

Cardiomyocyte apoptosis was assessed with another apoptosis detection kit (11684795910, Roche, USA) using an immunofluorescence method. After TUNEL staining and DAPI staining, cells were imaged by fluorescent microscopy using 488nm excitation and 530nm emission. Cells exhibiting green fluorescence were defined as TUNEL positive, apoptotic cells. A ratio of the apoptotic cell number to the total cardiac myocytes number was calculated as the apoptotic index in each experimental group.