(A-C) Whole-mount images of vasculature in the uvea (A), diaphragm (B), and trachea (C). Mice were intravenously injected with Alexa Fluor 594-conjugated anti-nepmucin mAb (red) and Alexa Fluor 647-conjugated anti-PV-1 mAb (blue), and transcardially perfused with 4% PFA. Collected tissues were observed using a laser-scanning confocal microscope, and Z-series images of each tissue were projected into one plane to show the vascular structures. (D, E) Mice were intravenously injected with Alexa Fluor 647-conjugated anti-nepmucin mAb (blue). After fixation, collected tissues were stained with Cy3-conjugated anti-α-smooth muscle actin mAb (red). The arterioles (D) and venules (E) were identified by the wrapping morphology of α-smooth muscle actin-expressing perivascular cells. Nepmucin-expressing vascular fragments enwrapped by α-smooth muscle actin+ cells are indicated by arrows. (F) A frozen section of tissue containing the ventral aorta and inferior vena cava was stained with Alexa Fluor 594-conjugated anti-nepmucin mAb (red) and FITC-conjugated anti-CD31 mAb (green). Note that nepmucin staining was absent in the aorta (AO) and vena cava (VC), whereas it was detectable in the capillaries of the surrounding adipose tissues. Ar: arteriole, Ve: venule. Scale bars, 100 µm.