4% Paraformaldehyde-fixed A375 (H) cell; Triton X-100 at r.t. for 20 min; Antibody incubation with (human CD63) monoclonal Antibody, unconjugated (bsm-30194M) 1:100, 90 min at 37°C; followed by conjugated Goat Anti-Mouse IgG antibody (green, bs-60296G-BF488) at 37°C for 90 min, DAPI (blue, C02-04002) was used to stain the cell nuclei. PBS instead of the primary antibody was used as the blank control.

The A375(H) cells were fixed with 4% PFA (10 min at r.t.) and then permeabilized with 90% ice-cold methanol for 20 min at -20℃,the cells then were incubated in 5%BSA to block non-specific protein-protein interactions (30 min at r.t.).Primary Antibody (green):Mouse Anti-human CD63 antibody (bsm-30194M): 1 μg/10^6 cells; Secondary Antibody (white blue): Goat anti-Mouse IgG-BF488(bs-60296G-BF488): 1 μg/test. Blank control (black): PBS. Acquisition of 20,000 events was performed.