2.8. Matrigel Plug Assay and Analysis of Hemoglobin Content

The effects of zileuton on angiogenesis in vivo were monitored using Matrigel plug assay by using growth factor-reduced Matrigel (in liquid at 4 °C). Prepared Matrigel (0.5 mL) containing 40 units/mL heparin and 10 ng/mL VEGF in the presence or absence of zileuton (50 µM) was injected into the anesthetized mice subcutaneously around the flanks area. Animals were randomly divided into 3 groups and administered the following: (a) PBS (used as a control); (b) VEGF (10 ng/mL) alone; (c) co-treatment of VEGF (10 ng/mL) and zileuton (50 µM). Mice were euthanized by CO₂ at day 7 for further analysis. Removed Matrigel plugs were fixed in 10% neutral-buffered formalin, processed for embedding in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). To quantitate the vascularization of the plug, the amount of hemoglobin (Hb) accumulated in the plug was measured using total Hb kits following the manufacturer’s protocol. Briefly, harvested plugs were weighted and incubated overnight in de-ionized water at 37 °C. Then, plugs were homogenized and centrifuged at 7000g for 15 min at 4 °C. Hb content was quantified by directly measuring the supernatants at OD405, calculated against as standard curve generated with purified porcine hemoglobin (Sigma-Aldrich, St. Louis, MO, USA), and normalized against wet weight of the plug.