*4.5. Immunohistochemistry*

Mice under deep anesthesia (ketamine and xylazine, 100 and 10 mg/kg, ip, respectively) were perfused with nitroprusside (10−<sup>4</sup> M) in PBS at ~100 mmHg with a reservoir for 3 minutes for maximal dilation and then with 2% PFA for 15 min for fixation. Brains were blocked with 10% goa<sup>t</sup> serum in 0.3% PBS-triton 1 hr at room temperature. Then the cortex area were incubated with 1:50 anti-eNOS rabbit IgG (sc-654, Santa Cruz Biotechnology, Santa Cruz, CA, USA); 1:100 anti-phospho eNOS rabbit IgG (ab75639, Abcam, Cambridge, MA, USA), 1:100 anti-acetylated-tubulin (T7451, Sigma), 1:100 anti-ZO1 (ab96587, Abcam), 1;400 anti-aSMA (Abcam, 32575, clone E184), overnight, 4 ◦C in a shaker, followed by incubation of 1:200 goa<sup>t</sup> anti rabbit-fluorescent conjugated with Alexa fluor ®568 (A10042, ThermoFisher Scientific, Grand Island, NY, USA) or Alexa fluor ®488 (A21208). Images of eNOS and phorpho-eNOS for pial collaterals and DMAs were taken using a Leica fluorescent stereomicroscope (Richmond, IL, USA), and the signal strength was measured using ImageJ (Bethesda, MD, USA). Endothelial cells were probed with Isolectin-GS-IB4-Alexa568 (I121415, ThermoFisher Scientific). Primary cilia (probed with anti-acetylated-tubulin antibody) in the lumen of pial collaterals were observed with a Zeiss 710 confocal microscope (Thornwood, NT, USA), with z stack scanning from the top of cortex down to a depth of ~30 um. Endothelial junctions (probed with ZO-1) were visualized with a Zeiss 710 confocal microscope.

#### *4.6. Collateral Primary Cilia and Endothelial Orientation Assessed by Scanning Electron Microscopy*

Mice were perfused with maximal vessel dilation as described above. Brain arterial vasculature was then casted using a Batson's No 17 Plastic Replica and Corrosion Kit (Polysciences, Inc, Warrington, PA, USA). Briefly, 1 ml of Batson's 17 was infused through the thoracic aorta. After fully curing, the brain tissue was removed using maceration solution, and the cerebral vasculature including the pial collateral regions were carefully persevered for emission scanning electron microscopy. The vasculature was observed under a Zeiss Supra 25 Field emission scanning electron microscope. Images of pial collateral and DMAs were saved for analysis of primary cilia and endothelial cell morphology. To measure the orientation of collateral endothelial cells, we use Photoshop to draw a line coordinate with the collateral axis and a second line coordinate with the endothelial cell axis (see Figure 2, panel C), and the angle formed was measured.
