P 30 gel exclusion column

Tissue was fixed in 4% PFA/PBS at room temperature for 10 minutes and equilibrated in 30% sucrose overnight at 4°C. Tissue was embedded in O.C.T. (TissueTek) and stored at −80°C. Sections were cut at 10 µm using a Leica CM3050S cryostat and incubated with primary antibodies overnight at 4°C. Primary antibodies were rabbit anti-TH (Abcam, ab112), chicken anti-GFP (Abcam, ab13970), rat anti-CD31 (BD Pharmingen, 553370), chicken anti-β-galactosidase (Abcam, ab9361), and mouse anti-smooth muscle actin (Sigma, A5228) used at 1:500. Mouse anti-smooth muscle actin antibody was directly conjugated to Cy5 NHS ester (GE Healthcare), and unbound dye was removed on a P-30 gel exclusion column (BioRad)³⁵ . Incubation with secondary antibodies was 45 minutes at room temperature. Secondary antibodies were conjugated to either Alexa Fluor 488, Alexa Fluor 555 (Life Technologies), or DyLight 488 (Jackson ImmunoResearch). Staining with 4’,6-diamidino-2-phenylindole (DAPI; 1 ng/ml, Life Technologies) was performed after incubation with secondary antibodies for 5 minutes at room temperature. Sections were mounted in Mowiol 4–88 (Polysciences) with DABCO (25 mg/ml, Sigma-Aldrich) and visualized on a Zeiss Axiophot fluorescence microscope. Tissue samples from three or more animals were stained for representative data shown (Fig. 1g, h, Fig. 3a, b, and Extended Data Fig. 3a–c, f–h).