TUNEL Assay

Apoptotic cells in myocardium were detected using a in situ cell death detection kit (Roche, 12156792910) following the manufacturer’s instructions. Heart sections were deparaffinized, hydrated, kept in permeabilization solution (0.1% Triton X-100) for 5 min, and incubated in TUNEL reaction mixture for 60 min at 37°C; the sections were then sealed in Prolong Gold Anti-Fade Reagent with 4-6-diamidino-2-phenylindole (DAPI, Invitrogen). Images were acquired via a fluorescence microscopy (Nikon) and quantified using ImageProPlus6.0 software (Media Cybernetics, Sarasota, FL, United States).