Immunostaining of P0 cultures and isolation and characterization of BM vascular endothelial cells. (Ai-vi) In situ staining of P0 cultures plated on fibronectin-coated chamber slides (LabTek; Nunc), cultured in EGM-2MV for 5 to 7 days. Vascular endothelial cells were identified by staining with a combination of VE-Cadherin–Alexa 488 and MECA32–Alexa 488 antibodies, and stromal cells were stained with rat anti–mouse PDGFRα/β (purified) antibodies and revealed with donkey anti–rat Cy3 and counterstained with DAPI. IgG2a and IgG1 isotypes were used for controls (Aiv-v). (B) Gating strategy for FACS purification of vascular endothelial cells from P0 cultures plated on fibronectin-coated 10-cm² dishes at 1 × 10⁶ mononuclear cells/cm² and cultured in EGM-2MV for 5 to 7 days and stained as described in “In situ staining.” (C) Phase-contrast images of Lin⁻CD105^BRIGHTPDGFRαβ⁻ cells at passage 3 and functional analysis of DiI-Ac-LDL uptake. (D) In situ staining of Lin⁻CD105^BRIGHTPDGFRαβ⁻ cells at passage 3 for endothelial markers, including VEGFR2 (Di), VE-Cadherin (Dii), CD31 and eNOS (Diii), MECA32 (Div), CD105 (Dv), and isotype controls (Dvi-vii). Nuclei were counterstained DAPI. Imaging was performed on an inverted fluorescence microscope (Olympus BX51) at both ×10 and ×40 (original magnification) and captured with an Olympus DP71 camera.