JAM-A^−/− and PECAM^−/− mice exhibit different profiles in terms of site of arrest of transmigrating leukocytes in IL-1β–stimulated cremaster muscle tissues. IL-1β–stimulated cremaster muscle tissues from WT, JAM-A^−/−, or PECAM-1^−/− mice were immunostained and prepared for analysis by confocal microscopy as detailed in “Immunofluorescence labeling and analysis of cremaster muscle tissues by confocal microscopy.” Briefly, tissues were immunostained for endothelial cell junctions using an anti–PECAM-1 mAb (in WT and JAM-A^−/− mice) or an anti–ICAM-2 mAb (in PECAM-1^−/− mice) (shown in red), the endothelial cell basement membrane using an anti–collagen IV Ab (shown in green), and neutrophils using an anti–MRP-14 mAb (shown in blue). The localization of neutrophils in relation to the endothelium and its basement membrane was determined and quantified by analyzing acquired three-dimensional images of whole blood vessels. (A) The number of neutrophils quantified to be at the level of the endothelium, arrested between endothelial cell junctions (left panel), or arrested between the endothelium and the endothelial cell basement membrane (right panel), in different strains of mice. Samples were observed using a Zeiss LSM 5 PASCAL confocal laser-scanning microscope, and the images were subsequently analyzed using Volocity three-dimensional rendering software. The number of leukocytes arrested within the venular walls was quantified in 200-μm sections (n = 5–12 cremaster muscle tissues per group). (B,C) Representative longitudinal (left panels) and cross-sectional (multiple small panels on the right) images of venules from JAM-A^−/− and PECAM^−/− mice, respectively. The left panels show three-dimensional images of venules stained for endothelial-cell junctions (red) and neutrophils (blue) only. The images on the right were obtained by cutting a cross-section (1-μm thick) of the venules on the left along the indicated dotted lines. The cross-sectional images corresponding to each of the numbered dotted lines are presented as a panel of 3 images in a row, showing the staining of endothelial-cell junctions (EC; red), neutrophils (PMN; blue), and the endothelial-cell basement membrane (BM; green). The arrows show the location of selected neutrophils, clearly indicating arrest of neutrophils at endothelial-cell junctions in the JAM-A^−/− mice and between the endothelium and the endothelial-cell basement membrane in the PECAM-1^−/− mice. Scale bar equals 10 μm. Statistically significant differences between WT and KO groups are shown by asterisks (***P < .001). Additional statistical comparisons are indicated by lines and number signs (##P < .01, ###P < .001).