Cell surface clustering of P-selectin and CD63 in stimulated HUVECs. (A) IL-4 treated HUVECs were grown to a confluent monolayer and stimulated with PMA followed by fixation. Coverslips were labeled with primary antibodies followed by secondaries conjugated to colloidal gold particles. Samples were then processed for scanning EM and distribution of gold particles analyzed. Labeling for P-selectin (10 nm gold) and CD63 (15 nm gold) shows a tendency of the 2 proteins to co-cluster. Panels i-iii show 3 example backscatter images of the gold labeling of P-selectin and CD63. Magnifications of example clusters are shown in panels iv-viii with gold particles shown in backscatter images (compo) and corresponding surface structures shown in secondary electron images (SEI). All scale bars represent 200 nm. (B) Quantification of scanning EM clusters, showing the percentage of clusters observed containing P-selectin, CD63 or both molecules together. Data were obtained from 5-10 separate low power images of equal magnification from 4 independent experiments. A total of 538 and 994 gold particles were counted for P-selectin and CD63 labeling, respectively. Data are shown as mean ± SD.