α sma cy3

The rat and mouse aortic rings were respectively cultured for 1 and 2 weeks before the staining. The rings were washed with PBS, fixed in 4% formaldehyde for 20 minutes. The wells were then washed once in PBS and the rings were permeabilized with 0.5% Triton X-100 in PBS for 30 minutes, before being washed twice in PBS. 100μl of BS-1 Lectin FITC (1 mg/ml; Sigma, cat. no. L9381/L5264) (1:200), anti-actin, α-SMA Cy3 (Sigma, cat. no. C6198) (1:500) or anti-NG2 (Millipore, ab5320) (1:200) was added and incubated overnight at 4°C. For IL-6Rα staining on aortic rings, 100μl of the unconjugated (1:200) IL-6Rα antibody was left overnight at 4°C. The following day the rings were washed with PBS and incubated with goat anti-rabbit Alexa 488 antibody (Life Technologies, A-11034) for 2 hours at room temperature. The plates were washed twice in PBS and the rings were removed from the 96 well plate, using a syringe needle, placed on a microscope slide and mounted with Prolong Gold DAPI containing medium (Invitrogen, cat. no. P36931). The slides were left to dry and imaged using confocal microscopy (Zeiss LSM 510 META).

Lungs were isolated, flushed with PBS, inflated and embedded with optimal cutting temperature compound, flash frozen in liquid nitrogen, and sectioned. Prior to staining lung sections were fixed in a solution of 4% paraformaldehyde (Electron Microscopy Sciences) and 0.01% Triton in PBS. Sections were stained with FITC conjugated rat anti-mouse CD31 (CD31-FITC, BD Biosciences, catalog #558738), Cy3 conjugated mouse monoclonal anti- α smooth muscle actin (αSMA-Cy3, Sigma-Aldrich, SKU C6198), and DAPI (Thermo Fisher Scientific). Lung sections from Tie2-Cre/YFP^fl/- transplanted animals were also stained with AF647 conjugated anti-GFP (GFP-AF647, Thermo Fisher Scientific, catalog #A-31852). Control specimens included unstained slides. Slides were labeled only with randomized animal identification number and data acquisition was performed without knowledge of the treatment group. The number of fully (>75% of vessel circumference) and partially muscularized αSMA-positive pulmonary arterioles (<100 μm diameter) were quantified from images taken using an Olympus microscope. In lung sections taken from Tie2-Cre/YFP^fl/- transplanted animals, pulmonary arterioles were further evaluated for the presence of cells expressing both YFP and CD31, identified as PACs (negative controls shown in Online Figure I).