Figure 1. VEGFR3 and intravital VEcadherin identify distinct immunophenotypic arteriole and sinusoidal BMEC niches.

Mice expressing a Vegfr3::YFP BAC transgene reporter were intravitally-labeled using an EC-specific antibody (VEcadherin). A) Representative flow cytometry contour plots of enzymatically-dissociated and hematopoietic lineage⁺ cell-depleted whole bone marrow, stained with antibodies raised against CD45, TER119, CD31, VEcadherin, and Endomucin. Gating strategies (dashed line) and percentage of parent populations are indicated. Note: VEGFR3^highVEcadherin^highCD31⁺ sinusoid bone marrow endothelial cells (BMECs) (green) and VEGFR3^low/−VEcadherin^lowCD31⁺ arteriole BMECs (red) can be distinguished using intravital VEcadherin staining in combination with hematopoietic exclusion (lineage⁻CD45⁻TER119⁻). B) Representative histogram of Endomucin stained VEGFR3^highVEcadherin^highCD31⁺ sinusoid BMECs (green) and VEGFR3^low/−VEcadherin^lowCD31⁺ arteriole BMECs (red). (C) Representative confocal images of femoral trabecular and diaphysis regions demonstrating VEGFR3⁻VEcadherin⁺CD31⁺Endomucin⁺ (yellow arrow) and VEGFR3^low/−VEcadherin⁺CD31⁺Endomucin⁻ (white arrow) arteriole BMECs. Scale bar = 100 μm.