Quantifying VEGF in Tumor Tissues

VEGF levels were estimated in plasma, tumor tissue homogenates by ELISA method. Tumors were homogenized in RIPA lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100) containing proteinase inhibitors (Complete Proteinase Inhibitor Cocktail Tablets, Roche, Indianapolis. IN). Tissue debris was pelleted and the resulting supernatant was used in subsequent analysis. Total protein concentration was determined with a BCA protein assay (Pierce, Rockford, IL). VEGF concentrations were determined according to the manufacturer's instructions. A minimum of 3 tumors per group were analyzed in duplicate [30] (link).