BM plug isolation and histologic assessment of intact vascular structures in BM plugs. (A) Representative images of denuded bones, removal of metaphysis, and isolated intact BM plug. (B-D) Resin-embedded sections of BM plugs (B,D) and remaining bone tissue (mid-diaphysis; C) after removal of marrow plug were sectioned as 5-μm-thick longitudinal sections and stained with hematoxylin and eosin, demonstrating intact vascular structures. (E) Whole-mount image of BM plug stained with a combination of the endothelial cell-reactive antibodies MECA32 and VE-Cadherin reveals a well-organized vascular reticulum throughout the marrow. BM plugs were stained with DRAQ5 to provide a nuclear counterstain and then immersed in prolong gold anti-fade mounting medium (Invitrogen). After applying a glass coverslip and sealing with nail hardener, specimens were inverted and allowed to cure overnight in the dark at room temperature before confocal imaging. Images were collected using 63× oil immersion objective of a Leica TCS SP5 confocal microscope and processed with the Leica LAS AF lite Version 2.6.7266 software. Z-stacked images were collected in 0.2-μm slices at depths of 15 to 25 μm with a pinhole of 1 (×63).