Immunofluorescence Microscopy Analysis Protocol

Immunofluorescence was performed as described previously [26 (link)]. Briefly, cells were harvested and allowed to attach for 24 h to cell culture dishes with glass bottoms (NEST Biotechnology Co., LTD.). After washing twice with PBS, the cells were fixed in paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, and blocked with 1% BSA in PBS for 1 h. The cells were first incubated with the indicated antibodies at 4 °C overnight, washed twice with PBS, and then incubated with the corresponding fluorescein-conjugated secondary antibodies for 1 h in the dark. Cell nuclei were stained with DAPI (Vector Labs). After washing, the cells were visualized using an A1R-A1 confocal laser microscope system (Nikon, Japan).