Immunofluorescence Microscopy of Cells

Experiments were performed as described previously 16 (link). Cells grown on chamber slides were fixed for 15 min with 4% paraformaldehyde, permeabilized for 10 min in phosphate-buffered saline (PBS) containing 0.2% TritonX-100, blocked for 1 h with 1% BSA and 0.5% goat serum in PBS, and then incubated with primary antibodies at 4°C overnight. After rinsing with PBS, the cells were incubated with secondary antibodies for 1 h at room temperature and the nuclei were stained with DAPI (Sigma) for 5 min. Alexa Fluor 555-conjugated goat anti-rabbit antibodies (Molecular Probes) were used as secondary antibodies. Following three washes with HBSS, fluorescence was examined by an Olympus Confocal Laser Scanning Microscope (OLYMPUS IX83-FV3000-OSR).