Morphologic analysis of collateral arteries in transparent mouse hindlimbs. Arteries were visualized by pigment perfusion in sham-operated (A) and ligated (B) hindlimbs. Diameter of collateral arterioles (A,B, ) was significantly increased (C, *P < .05 vs sham, n = 5) 7 days after ligation of the femoral artery. Immunofluorescence staining of ephrinB2 (Cy3) in collateral arterioles (D,F) was quantified by morphometry (L). In contrast to control conditions (D,L; *P < .05 vs sham, n = 4), endothelial cell ephrinB2 staining intensity (arrow) was significantly increased if the collateral arterioles underwent arteriogenic remodeling (F, arrow). CD31 immunofluorescence (Cy2) was used as a control to validate integrity of the endothelial cell monolayer (E,G; scale bars represent 10 μm). A species-matched control antibody did not produce any detectable immunostaining in collaterals from sham-operated (H, control antibody: red fluorescence; I, CD31: green fluorescence) or from ligated hindlimbs (J, control antibody; K, CD31; scale bars represent 10 μm). Individual collateral arterioles were isolated and subjected to RT-PCR analysis, which confirmed the increased expression of ephrinB2 in collateral arterioles undergoing arteriogenic remodeling (M, RT-PCR analysis of CD31 was used as an endothelial cell–specific internal standard).