Quantitative VEGF Expression Evaluation

VEGF expression levels were evaluated by an eight-year work experience pathologist using typical tumor samples collected from the patients. Immunostaining was performed using an anti-VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:100 in accordance with the manufacturer’s instructions. Briefly, formalin-fixed paraffin-embedded tissue sections were cut into 5-μm sections, which were then dried, dewaxed in xylene, rinsed in graded ethanol, and rehydrated in double-distilled water. Two pathologists who were blind to the clinical data scored the degree of staining. VEGF expression level was scored according to clinical practice: (−) represented no or rare expression (< 5% positive cells); (+) represented mild expression (6–25% positive cells); (++) represented moderate expression (26–50% positive cells); and (+++) represented strong expression (> 50% positive cells). Low VEGF expression was defined as VEGF (− and +), and high VEGF expression was defined as VEGF (++ and +++) [29 (link)].