(A) Diagram detailing the labeling method. Cremaster muscles of LysM-EGFP-ki mice were stimulated with IL-1β (50 ng for 2 hours) followed by an i.v. injection of biotinylated anti-Ly6G (2 μg) to label blood neutrophils at t = 90 minutes. The tissues were superfused with histamine (30 μM) or vehicle in conjunction with AF647-streptavidin (1 μg/mL) for 2 hours. (B) Representative confocal image of an IL-1β–stimulated postcapillary venule illustrating the extent of AF647-streptavidin labeling of neutrophils at different stages of trafficking (left panel). Right panels shown enlarged images of the boxed regions and illustrate examples of AF647-streptavidin^– luminal, AF647-streptavidin⁺ sub-EC, and AF647-streptavidin⁺ interstitial neutrophils. Scale bars: 5 μm. (C) Representative confocal IVM images of a tissue stimulated with IL-1β plus histamine (see Supplemental Video 6) illustrating the effective labeling of an rTEM event. The exemplified neutrophil shows that once the cell has breached an EC junction, the leading body part in the sub-EC space rapidly becomes AF647-streptavidin⁺ while the luminal body segment remains AF647-streptavidin^–. The AF647-streptavidin⁺ neutrophil can be easily tracked as it migrates in a reverse manner toward the vascular lumen and reenters the bloodstream. Luminal and cross-sectional views are shown with the arrows indicating the direction of motility of the indicated neutrophil. Scale bars: 3 μm. (D) Fluorescence intensity of AF647-streptavidin on neutrophils in the venular lumen, tissue, and cells exhibiting rTEM (n = 4 mice/group) during an IL-1β plus histamine reaction. Data are represented as mean ± SEM (each symbol represents 1 mouse/independent experiment). Statistically significant differences from luminal neutrophils are shown by **P < 0.01; ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post hoc test.