The anatomical position of aortic DCs in aortic wall. After immunostaining the aortic sheet using CD31 antibody to visualize the endothelium, confocal images were taken along the Z axis to visualize all dendritic processes from each EYFP⁺ DC and were reconstituted to a Z stack. (A) Reconstituted Z planes from aortic valve (top), aortic sinus (middle), and aortic arch (bottom) show that the aortic DCs are localized beneath the endothelium. A small number of DCs in the aortic sinus extend their processes into the vessel lumen (A, middle, arrow). (B) Three-dimensional images were reconstituted using the Imaris program to better visualize the position of aortic DCs. Arrows indicate the vessel lumen side. (C) Exposure of some dendritic processes into the lumen. The aortic sheets from EYFP transgenic mice were stained with CD11c antibody without tissue permeabilization to see whether dendritic processes could capture antibody from the lumen. The confocal images were reconstituted in three dimensions. (D) Biotinylated anti-CD11c and hamster IgG control antibody were injected i.v. to CD11c-EYFP mice. After 3 h, the aortic segments were isolated, incubated with horseradish peroxidase–conjugated streptavidin, and the stained CD11c was enhanced with Alexa Fluor 555 tyramide. (E) The elastic lamina was visualized using its autofluorescence. The confocal images were reconstituted in three dimensions. Each figure is representative of at least three experiments. Bars, 20 µm.