Apoptosis assessment using the TUNEL assay

In vivo TUNEL assays were performed to detect apoptotic cardiomyocytes induced by MI/RI (16,17). Myocardial tissue was fixed with 10% formaldehyde for 24 h, washed, dehydrated, and embedded in paraffin. TUNEL staining was carried out with an in situ Cell Death Detection Kit (cat. no. 11684817910; Roche Diagnostics) according to the manufacturer's instructions. Samples were added 50 µl TUNEL reaction mixture in the dark at 37°C for 60 min. Subsequently, the sections were counterstained with hematoxylin for a few seconds at 37°C. Five microscopic fields in each section were randomly selected to count the number of TUNEL-positive cells and total cells. An apoptosis index (AI) score was calculated as the ratio of apoptotic cardiomyocytes to total myocytes.