Proximity ligation assay (PLA) demonstrates P-selectin and CD63 interactions. (A) After fixation and blocking, HEK 293 cells transfected with wild-type P-selectin, tail-deficient mutant P-selectin or nontransfected (NT) cells were assayed for CD63-P–selectin interactions (surface and total) using the Duo-link II Probemaker kit after the manufacturers instructions. PLA signals and P-selectin expression levels were then detected using confocal microscopy. Images show representative confocal maximal projections of surface PLA signals and P-selectin. Scale bars represent 25 μm. (B) The mean number of surface and total PLA signals per cell were quantified using Volocity Version 5.3.1 software and this value was normalized according to the level of P-selectin expression in each cell. A total of 8-10 cells were analyzed for each condition. (C) CD63 siRNA or mock treated HUVECs were PMA-stimulated followed by fixation and blocking. Endothelial surface CD63–P-selectin interactions were assayed the same way. Images show representative confocal maximal projections with PLA signals in red and nuclei in blue. Scale bars represent 25 μm. (D) Quantification of panel C. Images were quantified by counting total number of PLA signals in each image and dividing by the number of nuclei in field of view. A total of 286 and 471 individual cells were counted for mock and CD63 KD samples, respectively taken from 3 independent experiments. ***P < .001 by Student t test. Data are shown as mean ± SD.