Intracellular localization of eNOS-GFP and phospho-eNOS in areas of the carotid artery experiencing different shear stress patterns after 2 days of cast placement. (A) In the top panel, phospho-eNOS in corresponding vascular regions visualized by en face immunofluorescence staining. Indicated are the low shear stress region (low), the high shear stress region (high), the oscillatory shear stress region (oscillatory), and the control region (undisturbed). Fluorescence was monitored by en face confocal microscopy in stacks of 500 × 500 μm each. In the middle panel, fluorescence of eNOS-GFP in endothelial cells located in the 3 different shear stress regions and in the contralateral carotid artery. In the bottom panel the top and middle panels are merged, showing the colocalization signal in yellow. (B-D) Quantification of the localization of (B) the phospho-eNOS, (C) the eNOS-GFP, and (D) the colocalization signals in response to the different shear stress fields in percentages of the total area (B-C) or of the total eNOS-GFP signal (D); n = 5, *P < .05 versus low shear stress. †P < .05 versus undisturbed shear stress. All data are means ± SEM.