The thin prep rat aortic ring assay: a modified method for the characterization of angiogenesis in whole mounts

The rat aortic ring model has gained broad acceptance as an angiogenic assay. This system can be used to study the activity of angiogenic and anti-angiogenic factors, and investigate the molecular mechanisms of the angiogenic process. We describe here a thin prep modification of the aortic ring model, which significantly simplifies the procedure and allows staining of aortic outgrowths as whole mounts. Using this procedure, intact preparations of angiogenic outgrowths are successfully and reproducibly stained with endothelial cell (anti-CD-31 and -Tie2 antibodies, Griffonia Simplicifolia isolectin-B4) and smooth muscle cell (anti-alpha-smooth muscle actin antibody) markers. Combined use of double immunostaining and confocal microscopy allows concurrent visualization of endothelial and mural cells in the same cultures. Whole mount immunostains of rat aorta cultures are an effective way to rapidly characterize the cellular composition of the angiogenic outgrowths, and localize proteins implicated in the regulation of angiogenesis. This method should facilitate the work of the many vascular biologists that have adopted the rat aorta model as a tool to study angiogenesis and its mechanisms.