Matrigel Plug Neovascularization Assay

The Matrigel plug neovascularization assay has been used in our previous publication [22 (link)]. In detail, mice were anesthetized by inhalation of 3% isoflurane and exposed for 10 min in this assay. Mice were injected subcutaneously with growth factor reduced (GFR) basement membrane matrix (Corning® Matrigel, 356231, Glendale, AZ, USA) containing 30 ng/mL VEGF (Peprotech, 100-20, Rocky Hill, CT, USA) and 50 U/mL heparin (Sigma-Aldrich, H3393, Darmstadt, Germany). The gel formed a solid plug as it reached the body temperature. After 14 days, plugs were collected and homogenized with 500 μL cell lysis buffer and centrifuged at 6000×g at 4 °C for 60 min. A colorimetric assay (Sigma-Aldrich, MAK115, Darmstadt, Germany) was used to detect hemoglobin with a microplate reader at 400 nm wavelength. The plug was harvested for histological and immunohistochemistry analysis.