Tcs sp5 2 cw sted

For clathrin-HC immunostaining, samples were fixed and permeabilized in methanol for 6 min at −20°C. For p230 immunostaining, samples were fixed with 4% formaldehyde in PBS for 20 min and permeabilized with 0.2% Triton X-100 in PBS for 30 min. Fixed cells were then blocked in 2% BSA in PBS for 30 min before antibody staining. Cells were then sequentially incubated for 1 hr at room temperature first with primary and then with secondary antibodies diluted in blocking buffer. Samples were analyzed with a confocal system (TCS SP5 II CW STED; Leica) in confocal mode using a 100x, 1.4 NA objective and HyD detectors (Leica). Alexa Fluor 488–, 568-, 594-conjugated secondary antibodies were used. Images were acquired using the Leica software and converted to TIFF files using ImageJ (version 1.43; National Institutes of Health). Two-channel colocalization analysis was performed using ImageJ, and the Pearson’s correlation coefficient was calculated using the Manders’ coefficients plugin developed at the Wright Cell Imaging Facility (Toronto, Ontario, Canada).