(A and B) Phorbol ester–treated ear tissues of WT and Prtn3^–/–Ela2^–/– mice were immunostained for laminin (LN; green) to visualize EBM and Gr-1 (red) to identify neutrophils. Lesions were examined 4 h after stimulus application by fluorescence microscopy as described in Methods. (A) Representative images of tissue from WT and Prtn3^–/–Ela2^–/– mice. Both genotypes developed strong and widespread neutrophil infiltrations. (B) Higher-magnification images of boxed regions in A. Prtn3^–/–Ela2^–/– neutrophils showed no retention at the EBM. Scale bars: 200 μm (A); 25 μm (B). (C) Overall neutrophil infiltrates were quantified as the percentage of Gr-1–positive cells per microscopic field. Data are mean ± SEM. Intravascular cells were excluded. No significant difference between WT and Prtn3^–/–Ela2^–/– mice was found (P = 0.63). In vitro migration of WT and Prtn3^–/–Ela2^–/– neutrophils directed by C5a through 3-dimensional collagen matrices was analyzed by time-lapse video microscopy (see Supplemental Video 1). (D) The tracks of WT (n = 41) and Prtn3^–/–Ela2^–/– (n = 42) neutrophils are shown, and the factor for directionality ± SEM is indicated. No impairment was observed regarding chemotactic directionality of Prtn3^–/–Ela2^–/– versus WT neutrophils (P = 0.19). (E) Velocities of single cells (individual points) were calculated and averaged (red bar). Prtn3^–/–Ela2^–/– neutrophils showed no significant difference versus WT cells (P = 0.30).