Figure 3. Analysis of mural cell vasoconstrictions in KO and WT mice.

(A) Mural cells labeled immunohistochemically with anti-NG2 antibodies, and stained with a DyLight 405 (blue) secondary antibody. (B) Mural cells labeled in vivo with 10 kD AlexaFluor 647 (red) fluorescent conjugated Dextran, injected intravenously 1–6 days before sacrificing the mouse for histological processing. (C) Composite image of panels A & B, showing double-labeling of mural cells. (D) Mural cells from panels A-C, now in the context of microvasculature labeled with both 2MD in vivo IV injected tetra-methyl rhodamine fluorescently-conjugated Dextran (yellow), and with antibodies against endoglin protein (vasculature endothelium) tagged with Alexa Fluor 488 (green) secondary antibodies. Notice that the vessels near the mural cells have strictures (arrows). (E) Traditional confocal stack from KO hippocampus of immunohistochemically labeled mural cells (α-SMA, red, with red arrowheads) and vessel strictures (white arrowheads), DAPI (blue) cellular nuclei, and 2MD fluorescein dextran labeled vessels (green). (F–H) 3D volumetric modeling of stack in panel E (panel F), progressively magnified and rotated (panels G-H) to reveal mural cell surrounding stricture of capillary vessel. See Video S7. (I) Stereological quantification of hippocampal capillary strictures is significantly greater for KO than WT animals (inset), with most strictures having a paired mural cell within 2 μM distance. Scales in panels A–D = 40 μM; E,F = 10 μM; G = 8 μM; H = 1.5 μM.