The gelatin sponge–chorioallantoic membrane assay

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Abstract
Here we present a method for the quantification of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane (CAM) based on the implantation of a gelatin sponge on the top of the growing CAM on day 8 of development. After implantation, the sponge is treated with a stimulator of blood vessel formation in the absence or presence of an angiogenesis inhibitor. On day 12, blood vessels that are growing into the sponge are counted at macroscopic and microscopic levels. The estimated timeline for carrying out this protocol is 10 d. The presence of a vascular network in the CAM requires a careful analysis to distinguish new capillaries from pre-existing ones. This limitation does not occur in the avascular cornea assay, which may also take advantage of different genetic backgrounds when carried out in transgenic or knockout mice. Nevertheless, the gelatin sponge–CAM assay is simple, inexpensive and suitable for large-scale screening. *Note: In the version of the article initially published online, references 6 and 7 were incorrect. The correct references are: 6. Serbedzija, G.N., Flynn, E. & Willet, C.E. Zebrafish angiogenesis: a new model for drug screening. Angiogenesis 3, 519–528 (2000). 7. Ribatti, D., Vacca, A., Roncali, L. & Dammacco, F. The chick embryo chorioallantoic membrane as a model for in vivo research on angiogenesis. Int. J. Dev. Biol. 40, 1189–1897 (1996). The error has been corrected in all versions of the article.