CD63 is enriched in microdomains on WPBs. Confocal images of a single fixed HUVEC immunolabeled with specific antibodies to CD63 (green) and VWF (red) (A) or expressing exogenous EGFP-CD63 (green) and immunolabeled for VWF (red) (B). Scale bars, 10 µm. Arrowheads indicate bright regions of CD63 (A) or EGFP-CD63 (B) closely associated with individual WPBs. Insets show, on expanded scales, the fluorescence, in grayscale, for VWF (left panels) and CD63 (middle panels) and the color merge image (right panels; VWF in red, CD63 in green) for WPBs indicated by a and b. (A-B) Images were taken at room temperature using a Leica SP2 confocal microscope (and software) equipped with a PL APO 100× 1.4NA objective. (Ci) Image from a live-cell confocal fluorescence experiment of an EGFP-CD63 (green) and VWFpp-mRFP (red) coexpressing HUVEC showing 2 WPBs containing discrete bright microdomains of EGFP-CD63 fluorescence. Intensity plots through the long axis of the upper WPB (white line) are shown in the line graph below (green: CD63, red VWFpp). (Cii) Histogram of the fold increase in mean EGFP fluorescence intensity in microdomains compared with nonmicrodomain regions (bulk WPB membrane) for 50 WPBs. Mean microdomain EGFP intensity was 2.5- ± 0.7-fold (n = 49 WPBs; range, 1.4-4.1) that in the bulk membrane of the corresponding WPB. (C) Images were taken at 37°C using a Leica SP5 with an HCX PL APO CS 100× 1.46NA oil objective, pinhole (airy) 1.5, zoom 30 to 35.5, scan speed 1400 Hz in xyt acquisition mode.