Migration of neutrophils through the pericyte sheath is site specific. (A) A representative 3D reconstructed image of TNF-stimulated cremasteric postcapillary venule from a αSMA-RFPcherry x Lys-EGFP-ki mouse (180 min after inflammation) illustrating the existence of sites in the venular wall that support the transmigration of multiple consecutive neutrophils (indicated by arrows). (B) Higher magnification images acquired at indicated time points illustrating the time course of migration of multiple neutrophils (labeled 1–6) through the same site within the pericyte layer (blue circles). The sequence of images initiates with the breaching of the pericyte layer by cell 1. (C and D) Two examples showing the ability of a neutrophil to exhibit hesitant transmigration through gaps in the pericyte sheath, i.e., exhibit multiple protrusions and/or oscillatory movements before finally breaching the pericyte layer from a TNF-stimulated cremaster muscle of an αSMA-RFPcherry x Lys-EGFP-ki mouse as observed by 4D confocal IVM. The top panels are images of a half cremasteric venule viewed from the luminal side (C) or extravascular space (D). The boxed regions show the transmigration of a neutrophil through the pericyte sheath at the indicated time points. The arrows indicate a neutrophil extending a protrusion toward the extravascular space that was then withdrawn once (C) or twice (D) after TEM before completing the extravasation response through the pericyte layer. All images are representative of at least n = 6 experiments (1 vessel/mouse, 1 mouse/experiment). Bar, 10 µm.