CaMKIIδ is required for neutrophil TEM in vivo. (A) iVE-Cre CaMKIIδ^fl/fl mice were injected for 5 d consecutively with either tamoxifen or corn oil as described in the Materials and methods. After allowing for a 1-wk recovery period, one ear of each mouse was treated topically with croton oil in an acetone:olive oil carrier while the other ear was treated topically with carrier alone. After 5 h, the mice were sacrificed and their ears processed for immunofluorescence imaging using confocal microscopy. Representative images from control and endothelial cell CaMKIIδ knockout mice are shown. Here, neutrophils, endothelial cell junctions, and basement membranes were visualized with antibodies against MRP14 (green), PECAM (red), and collagen IV (blue), respectively. Insets show the orthogonal view at the position denoted by the dashed line. Orthogonal inset 1 shows two leukocytes (i and ii) outside the blood vessel and basement membrane. Orthogonal insets 2 and 3 show three leukocytes arrested on the apical surface (iii–v). Scale bar is 10 µm. (B) Schematic of the neutrophil positional scoring system used to quantify the locations of neutrophils observed in A. (C) Quantitation of the total number of neutrophils per field. Only neutrophils within 50 µm of the vessel were scored. Data shown are the average and standard deviations of the three independent experiments. (D) Quantitation of the leukocyte positions from the images collected in A. Data shown are the average and standard deviations collected from three separate experiments. At least five fields with at least 100 neutrophils for each mouse were analyzed. Data shown do not include data for the neutrophils found in the luminal position. ** denotes P value <0.01 with Student’s t test. Carrier-only ears showed no signs of inflammation, and neutrophils were rarely found in the tissue or associated with the vessels (data not shown).