Cell surface P-Selectin clustering is reduced in CD63-depleted HUVECs. HUVECS, mock-treated or treated with 2 rounds of siRNA against CD63, were grown to a confluent monolayer. Cells were PMA-stimulated and fixed then coverslips labeled with primary antibodies against P-selectin followed by secondary antibodies conjugated to colloidal gold particles. Samples were then processed for scanning EM and distribution of gold particles then quantified. Scale bars represent 200 nm. (A) General views of cell surfaces indicating distribution of P-selectin. Large inserts are magnifications of the smaller boxes. Quantification reveals a reduction in the percentage of P-selectin found in clusters (B) and a reduction in the mean cluster size (C) after loss of CD63. Data were obtained from 5-10 separate low power images for each condition across 4 independent experiments. (B-C) ***P < .001 by Student t test. Data are shown as mean ± SD.