Ifluor 488 phalloidin

According to the manufacturer’s instructions, the TUNEL assay was employed to measure apoptosis in HEI-OC1 cells and cochlea culture explants (Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection; Invitrogen). DAPI (1:1000 dilution) was used to label all HEI-OC1 cells in the dark, while iFluor™ 488 phalloidin (1:1000 dilution; Yesen) in PBS for 30 min to visualize fluorescently labelled HCs. The coverslips were mounted and imaged using a laser scanning confocal microscope (Leica SP8; Leica, Germany) following treatment.

After culture, cochlea explants or HEI-OC1 cells on coverslips were fixed with 4% paraformaldehyde and blocked for 1 h at room temperature with PBT-1 (5% donkey serum, 0.1% Triton X-100, 1% bovine serum albumin and 0.02% sodium azide in PBS). The samples were then incubated overnight at 4℃ with primary antibodies diluted in blocking solution against cleaved-Caspase 3 (1:500 dilution; Cell Signaling Technology) and TIGAR (1:800 dilution; Abcam). The next day, the samples were incubated with secondary fluorescent antibodies (1:1000 dilution, Invitrogen) along with DAPI (1:1000 dilution, Sigma-Aldrich) in 0.1% Triton X-100 and 1% bovine serum albumin in PBS at room temperature for 1 h. The coverslips were mounted and imaged using a confocal laser scanning microscope (Leica SP8; Leica, Germany).
For the staining of phalloidin, after fixation, the basilar membranes on coverslips were incubated with iFluor™ 488 phalloidin (1:1000 dilution; Yesen) in PBS along with DAPI (1:1000 dilution) for 30 min in the dark. The coverslips were then mounted and confocal microscopy images were acquired.