Figure 3.

Extravasation of leukocytes into IL-1β-stimulated cremaster muscle is strongly reduced in VE-cadherin–α-catenin mice. (A, B) Confocal fluorescence microscopy was used to determine the number of extravasated neutrophils 3 h after intrascrotal IL-1β injection. (A) Representative images of whole mount stainings of cremaster labelled for PECAM-1 (green) and MRP-14 (red) to visualize endothelial cell contacts and neutrophils, respectively. Left panel: longitudinal vessel segment typically used for the 3D analysis and evaluation. Right panels: optical cross-sections of a venule depicting a neutrophil inside (upper right panel) and outside (lower right panel) the vessel. Projections of z-stacks taken on an LSM Zeiss 510 Meta, bar=20 μm. (B) Numbers of extravasated neutrophils in VEC–WT and VEC–α-C mice located within 50 μm next to the vessel were counted and are given per vessel segment (length 150 μm). A total of 60 (VEC–WT) and 66 (VEC–α-C) vessel segments from three mice of each group were analysed. (C–F) Intravital microscopy of neutrophil extravasation in venules of the cremaster muscle 4 h after intrascrotal administration of IL-1β. (C) Numbers of extravasated leukocytes in cremaster venules of VEC–WT and VEC–α-C mice per 1.5 × 10⁴ μm² tissue area determined by reflected light oblique transillumination microscopy. (D) Rolling flux fraction, (E) number of adhering leukocytes per 10³ μm² of venule surface area, (F) rolling velocity. A total of 28 or 35 vessels from 4 VEC–WT and 5 VEC–α-C mice were analysed. Haemodynamic parameters are given in Supplementary Table S6, showing that peripheral neutrophil counts, enhanced under inflammatory conditions, were similar in VEC–WT and VEC–α-C mice. ^***P⩽0.001; Mann Rank Sum Test.