Immunofluorescence for Cleaved Caspase 3 and TUNEL Assay

Cells were seeded in a confocal dish (Nest, China). After treatment, cells were fixated with 4% paraformaldehyde for 30 min, then permeabilized with 0.3% Triton X-100 for 15 min, blocked with 10% normal goat serum (Solable, China) for 1 h, incubated with cleaved caspase 3 (1:400, Cell Signaling Technology, 9664) at 4°C overnight. Next, Cells were washed with PBS and incubated with a fluorescent secondary antibody (1:1,000, goat anti-Rabbit IgG H&L Cy3, Abcam 6939) for 1 h at room temperature. Then the cells were stained with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) according to the manufacturer’s instructions of a TUNEL kit (Beyotime, C1086, China). Finally, nuclei were counterstained with DAPI.