Vesicles rarely co-localize with surface-associated clathrin. AF488-S470 vesicles (2.5 μg) were incubated with A549 cells for 1 h at 37°C. Cell surface was labeled using biotin and AF633-streptavidin (blue). Cells were washed, fixed, permeabilized, and probed with mouse anti-clathrin antibodies and AF555-labeled goat anti-mouse secondary antibody. Arrows indicate very occasional colocalization of clathrin and vesicle fluorescence at the cell surface.