4.8. In Vivo Angiogenesis (Matrigel Plug) Assay

The subcutaneous Matrigel plug assay in mice is the standard method for in vivo evaluation of the pro- and anti-angiogenic potential of different compounds. The assay was performed as described previously [52,53]. Briefly, Growth Factor Reduced Matrigel (Corning^®, Corning, NY, USA), thawed overnight at 4 °C, was mixed with either vehicle (control group), or with an equal amount of volume of AZ67 compound (0.115 mg/kg body weight, or 11.5 mg/kg body weight). Six hundred microliters of this mixture was injected subcutaneously into the flank area of 6-week-old C57BL/6 mice (Charles River, Wilmington, MA, USA), while under anesthesia. Two weeks after injection, mice were sacrificed, Matrigel plugs were removed and photographed using a dissection microscope to show the extent of vascularization. Next, Matrigel plugs underwent immunohistochemistry processing (fixation in 2% paraformaldehyde for 24 h, followed by paraffin embedding, sectioning and staining). Anti-CD31 antibody (77699S; Cell Signaling, Beverly, MA, USA) was used to delineate ECs. Sections were examined by light microscopy and photos were taken using an Olympus BX43 microscope (×5 magnification). CD31 positivity was quantified using the ImageJ Macro language. All animal procedures were approved by the ethical Committee of the University of Antwerp (protocol code 2017-81 and date of approval: November 2017).