Representative intravital micrographs showing specific accumulation of anti–VCAM-1 beads and control beads in nonirradiated BM.

Equivalent numbers of control and specific mAb-coated beads were injected into the carotid artery, and beads bound to the luminal surface of the vessel wall were counted 10 to 15 minutes later using appropriate filters. (A) Accumulation of control beads (YG) in nonirradiated BM microvessels. (B) Binding of anti–VCAM-1–coated beads (NR) in nonirradiated BM microvessels (same field of view), providing evidence for constitutive expression of VCAM-1 on BM ECs. Because this field was recorded through a filter set optimized for red (rhodamine) fluorescence, the autofluorescence of extravascular tissue was much lower than in panel A, which was recorded through an FITC filter set. Thus, the outline of BM microvessels is undetectable in panel B. (C) Computer-generated overlay of panels A and B. The ratio of specific/nonspecific bead binding in this experiment was 3.7. The variability in apparent size of different beads was due, in part, to the blurring of individual beads located out of the focal plane and, in part, because some signal bleeding occurred when using SIT cameras to record intensely fluorescent particles. All scenes were recorded through a × 20 water immersion objective (Zeiss).