Antibodies against CD99 and PECAM-1 trap neutrophils between endothelial cells while they transmigrate through endothelial monolayers in vitro. Bone marrow–derived mouse neutrophils were allowed to transmigrate through monolayers of bEnd.5 cells on laminin-coated transwell filters in the presence of antibodies against PECAM-1 (A-B), CD99 (C-D), or control antibodies (E-F). Endothelial cells were stained with cell tracker green, cell nuclei with Hoechst dye (blue), and at the end of the assay cells were fixed and neutrophils were stained with antibodies against CD11b (red). Cells were visualized by confocal microscopy, and each example is depicted as X-Y and underneath as X-Z presentation. Examples for neutrophils are shown that were trapped at a more apical position (panels A,C) or a more basal position (panels B,D) during diapedesis (en route) through the endothelial cell layer. Neutrophils varied between these 2 positions with similar frequency independent of whether antibodies against CD99 or PECAM-1 were used. Examples for neutrophils on top of endothelial cells (panel E) and embedded between endothelial cells, but not accessible for anti-CD11b staining (panel F) are shown. Quantification of neutrophils in the positions “en route” through the endothelial cell monolayer (G), between endothelial cells and not accessible for antibody staining (H), and in the bottom chamber of the transwell filters (I). **P < .01. *P < .05. Statistical analysis was done by ANOVA evaluating all available data with n ≥ 60 for co-IgG and n ≥ 90 for anti–PECAM-1 and anti-CD99.