Notch3 expression is restricted to vascular smooth muscle cells in human adult tissues. Notch3 expression was examined on paraffin sections from control individuals by in situ hybridization (a–l) and immunohistochemistry (m–r). Sections of myocardium (a–c), kidney (d–f), brain white matter (g–i), and renal artery (j–l) were hybridized with Notch3 antisense riboprobe (b, e, h, and k) and Notch3 sense riboprobe (c, f, i, and l). Kidney sections were hybridized with the HN3X riboprobes; all other sections with the SHB1 riboprobes. Bright fields (a, d, g, and j) and corresponding dark-field micrographs are shown. Notch3 is detected exclusively in the vessels (arrows) within the various parenchyma (a–i), and primarily in the smooth muscle cell layer of the media or in vessels of the adventitia within the renal artery (j–l). Small arrows in k indicate the internal elastica lamina, the thick arrow shows the external elastica lamina, and the star marks the luminal side. The bar in l represents 125 μm for a–c, 100 μm for d–f, 50 μm for g–i, and 200 μm for j–l. (m–r) Adjacent paraffin sections of brain frontal lobe were immunostained with primary antibodies recognizing the smooth muscle myosin heavy chain (mhc) (n), Notch3 extracellular epitopes 1E4 (o and q) and 2E11 (p), and the vascular endothelial cell marker CD34 (r). In m, primary antibody was omitted. Anti-Notch3 antibodies exclusively immunostained the smooth muscle cells (o–q) but not the endothelial cells (arrowheads). Bar, 100 μm for m–p and 20 μm for q and r (higher magnification of the boxed area of the vessel wall in o). Counterstaining was with toluidine blue (a–l) and with hematoxylin (m–r).