Fig. 5.

Number and size of VE-PTP particles in permeabilized and non-permeabilized aortic endothelial cells. a–a’’’’’ Confocal microscopic images of VE-PTP (red), Willebrand factor (vWF, blue or white), and VE-cadherin (green) in thoracic aorta endothelial cells with (a–a’’) or without (a’’’–a’’’’) permeabilization during staining for VE-PTP and vWF. VE-cadherin was stained in the presence of TritonX-100 in all specimens. Permeabilization was required for vWF staining in cytoplasmic organelles. Most small VE-PTP particles required permeabilization for staining, but the largest particles did not. Scale bar: 25 µm. b Area density measurements revealed significantly fewer ≤ 100-pixel VE-PTP particles but similar numbers of larger VE-PTP particles in aortas without permeabilization. b’ As expected, vWF staining required permeabilization, as almost none was found without TritonX-100. VE-cadherin values were similar in the two groups because TritonX-100 was used for VE-cadherin staining in all specimens (blue/red hashed bar). *P < 0.0001 by ANOVA or Student’s t test. c Measurements showed significantly fewer ≤ 10-pixel and 11 ≤ 100-pixel VE-PTP particles per endothelial cell without permeabilization. *P < 0.0001 by Student’s t test. d Line plots of aortas show significantly fewer 101 ≤ 200-pixel VE-PTP particles without permeabilization but similar numbers of > 200-pixel particles, consistent with a cytoplasmic location of most smaller VE-PTP particles and plasma membrane location of larger VE-PTP particles. P < 0.0001 by Kolmogorov-Smirnov 2-sample test. e Comparison of large VE-PTP particles shows significantly fewer 101 ≤ 200-pixel particles without permeabilization. Permeabilization had little effect on VE-PTP particles > 200 pixels, which fit with a plasma membrane location. *P < 0.0001 by two-way ANOVA. Mean ± SEM, n = 12 images from 6 mice/group