ICAM-2^−/−, JAM-A^−/−, and PECAM^−/− mice exhibit distinct profiles in terms of site of arrest of transmigrating leukocytes in IL-1β–stimulated cremaster muscle tissues. (A) A schematic diagram illustrating the quantification criteria used to analyze the position of leukocytes within fixed and immunostained whole-mounted cremaster muscle tissues. (B) WT, ICAM-2^−/−, JAM-A^−/−, or PECAM^−/− mice were given an intrascrotal injection of IL-1β (50 ng); and 4 hours later, the cremasters were dissected away from the mice, fixed, and immunostained to label neutrophils (MRP-14 with a 488-nm fluorochrome), EC (ICAM-2, JAM-A or PECAM-1 with a 555-nm fluorochrome), and venular basement membrane (collagen IV with a 633-nm fluorochrome), and prepared for analysis by confocal microscopy as detailed in “Immunofluorescence labeling and analysis of cremaster muscle tissues by confocal microscopy.” The localization of neutrophils in relation to the endothelium and its basement membrane according to the quantification criteria shown in panel A and detailed in text was determined and quantified by analyzing acquired 3-dimensional images of whole blood vessels as previously described.⁸  (C) The confocal images show representative longitudinal (top panels) and cross-sectional (bottom panels) images of IL-1β–stimulated venules from ICAM-2^−/−, JAM-A^−/−, and PECAM^−/− mice. The top panels show 3-dimensional images of venules stained for EC junctions (red represents EC) and neutrophils (blue represents PMN) only. The images below were obtained by observing a cross-section (1 μm thick) of the associated venules along the indicated dotted lines (numbered) showing the staining of EC junctions, neutrophils, and the EC basement membrane (green represents BM). The arrows show the location of selected neutrophils. Results are mean ± SEM from n = 4 to 8 mice (4 venules analyzed in each sample). Statistically significant differences between WT and knockout groups are shown as follows: *P < .05.