Anti αsma cy3

Following sacrifice, eyes were enucleated and fixed in 4% paraformaldehyde for 2 hours at 4°C. Retinas were dissected from the eye and incubated in blocking solution (0.5% Triton–X-100, 1% BSA in PBS) for 3 hours at room temperature. Retinas were stained with the following primary antibodies at 4°C overnight in PBLEC (1% Triton-X-100, 1 mM MgCl2, 1mM MnCl2, and 1mM CaCl2 in PBS [pH 6.8]); Biotinylated isolectin B₄ (1:50; Vector), anti-CD31 (1:100, BD Pharmingen), anti-NG2 (1:500, Millipore), anti-desmin (1:25, R&D), anti-αSMA-Cy3 (1:750, Sigma), anti-Notch3 (1:200, Santa Cruz), anti-collagen type IV (1:500, Cosmo. Bio), anti-laminin (1:500, Abcam), and 488-conjugated anti-GFP (1:200, Invitrogen). After washing with PBLEC, retinas were incubated with Alexa Fluor conjugated secondary antibodies (1:500, Invitrogen) in PBLEC. Stained retinas were flat mounted in 90% glycerol and confocal stacked images were acquired using a Nikon A1R microscope. Quantification of vessel density and pericyte coverage was performed using Image J on four 10× confocal stacked images per retina. Tip cells were defined as filopodia bursts and counted manually on four 40× images per retina.

Flow cytometry sorted bulk cells were resuspended in 100 µl PBS and spun for 2 min at 1000 rpm in a Cytospin 4 (Thermo Scientific). The cells were dried and fixed in 4% PFA for 10 min. After incubation in ice-cold acetone for 10 min and washing in PBS, all the following steps were performed in a humidified chamber. Unspecific binding sites were masked with serum-free blocking reagent (DAKO) for 90 min at room temperature. Rat-anti-PDGF-Rα (1:200, 14-1401-82, eBioscience) and anti-αSMA-Cy3 (1:100, C6198, Sigma Aldrich) were applied overnight at 4 °C. Goat-anti-rabbit-AF488 was applied 1:1000 in PBS + 1% BSA for 90 min at room temperature, followed by mounting with DAPI-containing mounting medium (Vector Laboratories). Fluorescent images were acquired with an Olympus BX63 microscope, DP80 camera, and cellSens Dimension v 1.12 software (Olympus Cooperation).

Whole-mount retina immunohistochemistry was performed as previously described in Kim et al., (2011) (link). Briefly, eyes were extracted and fixed in 4% paraformaldehyde for 10 min at room temperature. Retinas were dissected in PBS and post-fixed in 4% paraformaldehyde overnight at 4°C. Retinas were then permeabilized in PBS, 1% BSA, and 0.5% Triton X-100 at 4°C overnight, washed 2× for 5 min in 1% PBT (1% Triton X-100 in PBS), and incubated in Isolectin GS-IB4 (1:200, #I21411 Life Technologies, Grand Island, NY) and anti-αSMA Cy3 (1:100, #C6198 Sigma-Aldrich, Natick, MA) in 1% PBT overnight at 4°C. Retinas were washed 3× for 5 min and flat-mounted using ProLong Gold antifade reagent (#P36934 Molecular Probes, Eugene, OR). Flat-mounted retinas were analyzed by fluorescence microscopy using a Nikon Eclipe 80i microscope equipped with a Nikon DS-2 digital camera and by confocal laser scanning microscopy using an Olympus FV1000 confocal microscope. Quantification was performed using MetaMorph Image Analysis Software and ImageJ. At least four retinal leaves were quantified per animal to determine the vascular extension ratio, both eyes were examined in each animal for artery number, and three representative images were quantified from each animal for vascular coverage (representing the total isolectin-positive pixel area per image).