2.5. Immunofluorescence Assay

Cells were fixed in 3%–4% paraformaldehyde, permeabilized with 0.2% Triton X-100 for 10 min, and blocked with fish skin gelatin (FSG) blocking buffer (0.4% FSG, 0.05% Triton X-100) for 40 min at room temperature. The cells were then incubated with specific primary antibodies (0.5 ug) in 0.2% FSG/0.05% Triton X-100) overnight at 4 °C, washed with PBS, and incubated with Alexa Fluor conjugated secondary antibodies (0.2% FSG/0.05% Triton X-100) for 1 h at 37 °C. Nuclear staining was performed using TO-PRO3/PBS in PBS for 1 min. Cells were visualized and imaged using a confocal laser-scanning microscope (Carl Zeiss, Inc., San Diego, CA, USA) and processed with ZEN imaging software (Carl Zeiss, Inc.).