TUNEL assay

PC12 cells were fixed in PBS with 4% polyformaldehyde at room temperature for 15 min. The cells were washed once with PBS and then, apoptosis of PC12 cells in the different groups was detected by TUNEL via a in situ Cell Death Detection kit (Roche Applied Science). The kit was used in accordance with the manufacturer's protocol, and the ratio of apoptotic to total cells was calculated under fluorescence microscopy by observing three fields of view (magnification, x400). After that, ImageJ software (version 1.53, National Institutes of Health) was used to quantify the apoptosis rate.