TUNEL assay

The cell apoptosis was assessed using the terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) staining in accordance with the manufacturer’s instructions. After dehydration by ethanol, TUNEL reaction mixture was added and incubated with cells for 1 h at 37°C. The residual liquid was removed via washing with PBS. The cells were stained using 3,3′-diaminobenzidine (DAB) as a substrate for the peroxidase at room temperature for 10 min. For each section, ten different fields were randomly selected for counting at least 150 cells from at least three separate experiments. The number of TUNEL-positive cells was analyzed using light microscope system at 400× magnification in a blinder manner. Positive apoptotic cells were stained claybank [23].