TUNEL Assays

Apoptosis levels were determined by the TUNEL assay using the One Step TUNEL Apoptosis Assay kit (Dalian Meilun Biotechnology Co., LTD, China) according to the manufacturer protocol. Myocardial tissue sections were baked in an oven at 60°C for 30 min, deparaffinized with xylene (5 min, 3 times) and dehydrated with 100%, 95%, and 70% ethanol 3 times each. Next, the sections were incubated with protein kinase K for 30 min. After rinsing with Phosphate-Buffered Saline (PBS), TUNEL detection solution (including TdT and fluorescein-dUTP) was added for reaction at 37°C for 1 h. After washing with PBS 5 times, 1 μg/ml of DAPI the samples were incubated in the dark for 10 min at 37°C. Finally, tissues were washed with PBS and sealed with antifade solution (Solarbio, Beijing, China) and then observed with an Ortho fluorescence microscope (DS-Ri2, Nikon, Japan). Cells with green nuclei were deemed positive for apoptosis. Five representative fields were randomly selected at ×200 magnification. The apoptosis rate was calculated as the ratio of TUNEL positive cardiomyocytes nuclei to the total number of cardiomyocytes nuclei (n = 5).