Intense and granular appearance of Notch3 immunoreactivity in CADASIL brains. (a–h) Paraffin sections from control (C1–C3; a, c, e, and g) and CADASIL brains (cad1–cad4; b, d, f, and h) including sections from white matter (a and b), cortex (c, d, g, and h), and brainstem (e and f), were probed with the 1E4 anti-Notch3 antibody. The examples shown are from 3 control individuals and 4 CADASIL brains matched for formalin fixation conditions and duration: 48 hours for control 1 (a and g) and CADASIL patient 1 (b); 0.5 months and 3 months for control 2 (c) and CADASIL patient 2 (d), respectively; 8 years for control 3 (e) and CADASIL patient 3 (f), and 1 year for CADASIL patient 4 (h). Notch3 immunoreactivity is much more intense within smooth muscle cells of vessels from CADASIL brains than in control brains (a–h, right column compared with left column) for all durations of fixation. Granular and intense Notch3 immunostaining is also obvious within pericytes of capillaries (arrows, nuclei of pericytes) (h). Identical observations were obtained in brains from other donors (4 CADASIL patients, 2 controls). Bar, 50 μm for a–f and 20 μm for g and h. (i–l) Adjacent paraffin sections from CADASIL brain (patient 1) were probed with the 2E11 anti-Notch3 antibody (l), the anti-CD34 antibody (which is of the same isotype as the 1E4 and 2E11 antibodies) to identify the endothelial cells (j), the anti–smooth muscle α-actin antibody (to identify smooth muscle cells) (k), or without primary antibody (i). Bar, 50 μm. Counterstaining was with hematoxylin.