Retinal Vascularization Dynamics Analysis

Gene deletion was induced by intragastric injections to pups with 50 μg tamoxifen (1 mg/ml) at P0, P1 and P2. Mice were sacrificed at P5 for analysis of retinal vasculature as previously described32 (link). The retinas were incubated with isolectin B4 (IB4) and the following antibodies: anti-Collagen IV (Millipore, #AB769), anti-Erg1/2/3 (Santa Cruz, #SC353), anti-Phospho-Histone H3 (PH3, Millipore, #06-570). Retinas were imaged using a Nikon 80i fluorescence microscope and a Leica SP5 confocal microscope with a Leica spectral detection system (Leica 15 SP detector) and the Leica application suite advanced fluorescence (LAS-AF) software. Quantification of retinal vascular development and immunostaining were done using the Biologic CMM Analyser Software and ImageJ.

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Yu P., Wilhelm K., Dubrac A., Tung J.K., Alves T.C., Fang J.S., Xie Y., Zhu J., Chen Z., De Smet F., Zhang J., Jin S.W., Sun L., Sun H., Kibbey R.G., Hirschi K.K., Hay N., Carmeliet P., Chittenden T.W., Eichmann A., Potente M, & Simons M. (2017). FGF-dependent metabolic control of vascular development. Nature, 545(7653), 224-228.

Publication 2017

anti-Erg1/2/3 anti-Phospho-Histone H3 ( Leica 15 SP detector CMM Analyser Software

Antibodies Biologic Collagen iv Confocal microscope Erg1 Fluorescence Fluorescence microscope Gene deletion Histone h3 Isolectin Mice Retinal vascular Retinal vasculature Retinas Tamoxifen

Corresponding Organization : Yale University

Other organizations : Max Planck Institute for Heart and Lung Research, Dana-Farber Cancer Institute, KU Leuven, Gwangju Institute of Science and Technology, Sinotech Genomics (China), University of Illinois at Chicago, VIB-KU Leuven Center for Cancer Biology, Paris Cardiovascular Research Center

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Variable analysis

independent variables

Tamoxifen injections (50 μg, 1 mg/ml) at P0, P1, and P2

dependent variables

Retinal vascular development
Immunostaining of retinal cells (Collagen IV, Erg1/2/3, Phospho-Histone H3)

control variables

Age of mice (sacrificed at P5)
Experimental procedures (as previously described in reference 32)

controls

No positive or negative controls were explicitly mentioned.

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