Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at 4°C and pre-treated with 10% normal goat serum (NGS, Calbiochem) in PBS with 0.04% Triton X-100 (PBS-T) for 1 hour at room temperature (RT) to prevent unspecific antibody-binding. Then cells were incubated with the respective primary antibodies in 2% NGS in PBS-T for 1 hour at RT (rabbit anti-CNIH-2, 1∶250, [26]; mouse anti-GM130, 1∶100, BD Transduction Laboratories; chicken anti-MAP2, 1∶10.000, Abcam). Immunoreactivity was finally visualized by secondary anti-mouse, anti-rabbit and anti-chicken antibodies conjugated to cy-2, cy-3, or cy-5 (1∶250 in 10% NGS in PBS-T, Dianova).
