Abstract
The retina is a powerful experimental system for the analysis of angiogenic blood vessel growth in the postnatal organisms. The three-dimensional architecture of the vessel network and processes as diverse as endothelial cell (EC) proliferation, sprouting, perivascular cell recruitment, vessel remodeling or maturation can be investigated at high resolution. The characterization of physiological and pathological angiogenic processes in mice has been greatly facilitated by inducible and cell type–specific loss-of-function and gain-of-function genetics. In this paper, we provide a detailed protocol for tamoxifen-inducible gene deletion in neonatal mice, as well as for retina dissection, whole-mount immunostaining and the quantitation of EC sprouting and proliferation. These methods have been optimized by our laboratory and yield reliable results. The entire protocol takes ~10 d to complete.
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Acknowledgements
We thank S.F. Rocha for her help, A. Eichmann for sharing unpublished data, S. Volkery for microscopy and M.L. Bocheneck for reading the paper. The Max Planck Society and the University of Münster have provided support and funding for this research.
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M.E.P. and R.H.A. designed the experiments; M.E.P. and I.S. carried out the experiments; M.E.P., R.B. and R.H.A. contributed to the development of the methods and wrote the paper.
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Supplementary information
Supplementary Video 1
Intragastric injection of tamoxifen in a one–day–old mouse. (MOV 6883 kb)
Supplementary Video 2
Retina dissection. (MOV 9397 kb)
Supplementary Video 3
Retina dissection. (MOV 5141 kb)
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Pitulescu, M., Schmidt, I., Benedito, R. et al. Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice. Nat Protoc 5, 1518–1534 (2010). https://doi.org/10.1038/nprot.2010.113
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DOI: https://doi.org/10.1038/nprot.2010.113
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