Cy3 αsma

Angpt4^Cre; Rosa26^mT/mG mice eyes were fixed in 4% PFA, retinas were dissected and flattened in Immu-Mount (Thermo Scientific) between cover and objective glass for microscopy analysis. For retina immunofluorescent staining, dissected retinas were treated for 1 hr with cold methanol, permeabilized and blocked for 2 hr with 50% BSA–1% Triton–1xPBS. Primary antibody stainings were carried out overnight using Cy3-αSMA (C6198, Sigma-Aldrich), SM22 alpha (ab10135, Abcam), ColIV (AB756P, Merck Millipore), GFAP (ab7260, Abcam), pimonidazole (Pab2627, Hypoxyprobe) and biotinylated isolectin B4 (B-1205, Vector) antibodies, and visualized with Alexa Fluor 488 streptavidin, Alexa Fluor 488-, Alexa Fluor 647- and Cy3-conjugated secondary antibodies (Jackson ImmunoResearch). Retinas were imaged using Olympus FluoView FV1000 or Zeiss LSM780 confocal microscope. Coverage of αSMA-positive cells was quantified from branching point region of two largest veins at peripheral area of retina. The width of the veins was measured just below the venal branching point at peripheral retina or close to optic nerve head for comparison. 5-Bromo-4-chloro-3-indolyl β-D-galactosidase (X-Gal) staining of retinas was performed as described in Gossler and Zachgo, 1994 .