CD63 in WPB ILVs is of endosomal origin. (A) Confocal fluorescence image of HUVECs incubated live in the presence of an extracellular TRITC-conjugated mouse anti-CD63 antibody (red; 1:55 dilution; 24 hours). The cells were fixed and immunolabeled with a specific antibody to VWFpp (green). Scale bar, 20 µm. The region in the white box is shown as grayscale insets to illustrate the accumulation of extracellularly applied TRITC–anti-CD63 in WPBs. Examples of the accumulation of extracellularly applied TRITC-anti-CD63 in microdomains in WPBs in control (B; mock transfected) or EGFP-CD63–transfected (C) HUVECs incubated live with the TRITC–anti-CD63 antibody (red) and subsequently immunolabeled for VWFpp (blue) and EGFP-CD63 (anti-GFP antibody, green). TRITC–anti-CD63 can be seen in bright microdomains on WPBs that colocalized with EGFP-CD63 microdomains. Scale bars, 2 µm.