Immunofluorescence staining

Immunofluorescence staining of the cells was performed as previously described (23) with some modifications. The cells were cultured on coverslips for 48 h prior to fixation. The cells were then fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature. Following 3 washes in PBS containing 50 mM NH₄Cl, the cells were permeabilized using 0.1% Triton X-100 in PBS for 5 min. After washing in PBS, the cells were soaked in blocking solution (PBS containing 5% fetal calf serum) for 15 min, and then incubated for 30 min with primary antibodies diluted in blocking solution. After washing 3 times in PBS, the cells were incubated with rhodamine- or fluorescein-conjugated secondary antibodies. The cells were analyzed as previously described (24) using an Olympus microscope.