PECAM-1 distribution on cultured HUVECs during PMN transmigration.

(A-D) Specimens were fixed with 4% paraformaldehyde in the presence of 0.5% Triton-X 100 and subsequently immunostained with anti–PECAM-1 antibody and fluorescence-conjugated secondary antibody. (E,F) Specimens were prelabeled with the primary antibody against PECAM-1, fixed, and finally labeled with secondary antibody. Confocal fluorescence micrographs B, D, and F were taken from the same specimens from which phase-contrast micrographs A, C, and E, respectively, were taken. Triangle symbols point at the stain-free areas, which correspond to the PMNs' pseudopodia that were penetrating the endothelial monolayer. Note: 2 pseudopodia from the same PMN might simultaneously penetrate the endothelial monolayer (C-D).