Inhibiting CaMKII in vivo significantly reduces neutrophil TEM. (A) iVE-Cre CaMKIIN^fl/fl mice were injected for 5 d consecutively with either tamoxifen or corn oil (control) 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 were processed for immunofluorescence imaging using confocal microscopy on whole-mount specimens. Representative images from control and CaMKIIN-expressing (tamoxifen-injected) mice are shown. Neutrophils and endothelial cell junctions were visualized with antibodies against MRP14 (blue) and PECAM (red), respectively. Insets show the orthogonal view at the position denoted by the dashed line. Orthogonal inset 1 shows three leukocytes (i–iii) outside the blood vessel. Orthogonal insets 2 and 3 show two leukocytes arrested on the apical surface (iv and 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) Quantification of the total number of neutrophils per field. Data shown are the average and standard deviations of the three independent experiments. Only neutrophils within 50 µm of the vessel were scored. (D) Quantification of the leukocyte positions from the images collected in A. Data shown are the average and standard deviations 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).