Quantification of Cardiac Apoptosis by TUNEL

In brief, the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method was employed using the commercially available in situ Cell Death Detection Kit, Fluorescein (11684795910, Roche, Mannheim, Germany) to quantify the apoptosis in cardiac tissue at the 24-h time point. Sections were mounted with UltraCruz Aqueous Mounting Medium with DAPI (Sc-24941; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and images were taken at 20× using IRIS digital imaging system (Logos Biosystems, Annandale, VA, USA). For the quantification of TUNEL-positive cells, approximately 8 fields per section were examined and expressed in the percentage of TUNEL-positive cells relative to the total cell number (nuclei) [16 (link),19 (link)].