In vivo Angiogenesis Assay in Mice

In vivo angiogenesis experiments were performed as previously described by Malinda (2009) (link). A mixture of basement membrane matrix (ice-cold phenol red-free, reduced growth factor, Thermo Fisher Scientific) and conditioned medium (0.5 mL, 9:1 proportion) was injected subcutaneously into two-months-old C57Bl/6 wild-type mice (n = 4). Each mouse received two implants, totaling eight plugs per group. A buffered saline was included as a negative control during the assay. After 7 days, the mice were euthanized and the plugs were excised, photographed, and processed. Quantification of blood vessels was achieved using immunofluorescent visualization of blood vessels on frozen Matrigel sections and by measuring the amount of hemoglobin (Hb) contained in the plugs. Frozen Matrigel sections were stained with rat anti-mouse CD31 antibody (R&D system, 0.125 μg/mL in BSA/normal serum solution) followed by incubation with Alexa 488-conjugated anti-rat antibody (Molecular Probes, 10 μg/mL in PBS) as described previously by Ribeiro et al. (2019) (link). For quantitation of functional vessels formed, the plugs were homogenized in distilled water and centrifuged at 2,400 × g for 5 min. The supernatant was mixed with Drabkin’s reagent (Sigma-Aldrich) for measurement of Hb. After 15 min at room temperature, the absorbance of the mixture was measured at 540 nm.