Cultures were washed with PBS and fixed using 4% paraformaldehyde (no. 15714, Electron Microscopy Sciences) for 20 min, then washed three times with PBS 1× plus 0.01% Triton X-100 prior to preincubation with blocking solution made of PBS 1× plus 0.01% Triton X-100 and 10% donkey serum (Sigma-Aldrich), for at least 1 hr. Cultures were then incubated in primary antibody solution overnight at 4°C, followed by an incubation with secondary antibodies for 1 hr at room temperature. See Supplemental Experimental Procedures for antibody details and confocal microscopy.
