CD99 gene inactivation affects neutrophil extravasation at 2 steps. Three-dimensional (3D) confocal image analysis of cremaster was used to determine sites of arrest of transmigrating leukocytes in WT and CD99^−/− mice. Mice were injected intrascrotally with interleukin-1β (50 ng) for 4 hours, cremaster muscles were prepared from the sacrificed animals, and whole mounts were immunostained for laminin α5 (green), ESAM (purple), and MRP14 (red) to stain the basement membrane, endothelial cells, and leukocytes, respectively. (A) Representative image of a blood vessel used for 3D analysis and evaluation. The first 3 panels show single stainings for the basement membrane (green), endothelium (purple), and leukocyte (red), only, and the last panel shows the merger of all 3 channels. Bars represent 25 µm. (B) Schematic drawing illustrating the criteria used to delineate the 3 positions in which leukocytes are found during extravasation. Position A includes leukocytes that are adhered to the endothelium and usually appear round, position B includes leukocytes which are trapped between the endothelium and the underlying basement membrane and appear flat and oriented parallel to the blood vessel, and position C includes cells which have transmigrated into the tissue. (C) Representative cross-sections of vessels with leukocytes in the 3 positions. (D) Graph presenting the absolute numbers of leukocytes per vessel segment in each of the 3 positions. Data are shown as mean ± standard error of the mean (n = 51 vessels for CD99^−/− and n = 52 vessels for WT mice). **P < .01 and ***P < .01, as per Mann-Whitney U test.