Immunohistochemical staining

Formalin-fixed, paraffin-embedded tissue specimens were cut into 7-μm-thick sections. The VEGF and VEGFR-2 proteins in the primary lesions were detected by immunohistochemistry. All primary antibodies were purchased from Abcam (Cambridge, UK). The mouse and rabbit monoclonal antibodies were purchased from Sener Biotechnology (Shanghai, China). The positive controls were obtained from known positive cases of GC, while the negative controls were subjected to the same procedure except that the primary antibody was replaced by PBS.

Two pathologists evaluated the slides independently. The immunoreactivity of VEGF was evaluated as follows: a product of the intensity and percentage of positive cells of > 6 was defined as positive. The scores for positive immunohistochemical staining were 0–20%, 1; 21%-40%, 2; 41–60%, 3; 61–80, 4; and 80–100%, 5. The color strength score was defined as follows: no coloration, 0; light brown (weak), 1; brown (medium), 2; and dark brown (strong), 3.⁹ The immunoreactivity of VEGFR-2 was evaluated as follows: a product of intensity and the percentage of positive cells of ≤ 1 was defined as negative, 2–4 was defined as weakly positive, and ≥ 5 was defined as strongly positive. The scores for positive immunohistochemical staining were < 5%, 0; 6–25%, 1; 26–50%, 2; 51–75, 3; and 76–100%, 4. The color strength score was defined as follows: no coloration, 0; light brown (weak), 1; brown (medium), 2; dark brown (strong), 3.¹⁰