Cells were fixed in freshly prepared 4% paraformaldehyde solution for 15 min, washed three times with phosphate-buffered saline (PBS) and then treated with 0.25% Triton X-100/PBS for 5 min. The fixed preparations were blocked in 3% BSA/PBS for 1 h, incubated with primary antibodies for 1 h at room temperature, washed three times with PBS and incubated with Alexa Fluor 555 or Alexa Fluor 488 conjugated secondary antibody for another 1 h (Invitrogen). Fluorescent images were acquired by confocal microscopy.