Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

Apoptotic cells were stained using a TUNEL kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. BEAS-2B cells were fixed with 4% paraformaldehyde for 30 minutes and then incubated with PBS containing 0.3% Triton X-100 for 5 minutes. The samples were incubated in PBS containing 0.3% hydrogen peroxide at room temperature for 20 minutes to inactivate endogenous peroxidase. Sections were then three times with PBS. The samples were incubated for 60 minutes in the dark with 50 µL of biotin-labeled solution, and for 30 minutes at room temperature with 50 µL streptavidin-horseradish peroxidase solution. 3,3ʹ-Diaminobenzidine was used as a substrate. Nuclei were stained with hematoxylin. Staining was observed under a fluorescence microscope.