We present a simplified method for conducting aortic ring assays which yields robust sprouting and high reproducibility targeted towards matrix biologists studying angiogenesis and extracellular matrix signaling. Main adjustments from previously established protocols include embedding aortic rings between two layers of 3D type I collagen matrix and supplementing with vascular endothelial media. We also introduce a concise and effective staining protocol for obtaining high-resolution images of intracellular and extracellular matrix proteins along with a more accurate protocol to quantify angiogenesis. Importantly, we present a novel method to perform biochemical analyses of vessel sprouting without contamination from the aortic ring itself. Overall, our refined method enables detection of low abundance and phosphorylated proteins and provides a straightforward ex vivo angiogenic assay that can be easily reproduced by those in the matrix biology field.
Keywords: Aortic rings; Collagen; DAPI, 4′,6-diamidine-2′-phenylindole dihydrochloride; ECM, extracellular matrix; Endothelial cell markers; Extracellular matrix; HA, hyaluronan; HABP, HA-binding protein; Hyaluronan binding protein; IB4, Griffonia simplicifolia isolectin B4; PBS, phosphate buffered saline; PERK, protein kinase R-like endoplasmic reticulum kinase; PFA, paraformaldehyde; RIPA buffer, radioimmunoprecipitation assay buffer; Sprouts.
© 2020 The Authors.