Immunohistochemistry (IHC) for the detection of VEGF and micro-vessel density (MVD)

Immunohistochemistry was used to detect VEGF in mice tumor tissues. Tumor MVD was measured with CD31 as the positive index. Rabbit anti-human polyclonal antibodies VEGF, CD31 (Upstate, USA) and the EnVisionTM detection kit (DAKO, Denmark) were used for staining. Paraffin-embedded sections (3 um) were deparaffinized, hydrated, incubated at room temperature for 10 min and then washed in PBS for 10 min. After microwave antigen repair, sections were reacted in non-immune animal serum at room temperature for 10 min. Following addition of the first antibody (1:200), the section was placed in a wet incubator box at 37°C for 60 min. Sections were washed in PBS for 10 min, incubated using the EnVisionTM kit for 10 min, washed again in PBS for 10 min, incubated in the substrate-chromogen solution for 10 min and washed in distilled water. Sections then underwent DAB coloring, hematoxylin counterstaining and mounting with neutral gum. The primary antibody was replaced with PBS in negative controls. Under the microscope, we selected five different visual areas under high power, with a total of 100 cells. VEGF-positive cells presented granular yellow. Cell positivity was scored as follows: <5% positive cells = 0; 5 ∼15% = 1; 15∼50% = 2; > 50% = 3. Staining intensity was scored as follows: positive cells were uncolored = 0; yellow = 1; brownish yellow = 2; brown = 3. Final scores were calculated as the percentage of positive cells multiplied by staining intensity score: negative (−): 0–2; weakly positive (+): 3–5; positive (++): 6–8; strongly positive (+++): 9–12. CD31-positive cells in tumor tissues presented brown or brownish yellow and represented endothelial cells and tumor neovascularization. We identified “hot spots” (cancer nest peripheral vascular dense area) via light microscopy at 40 × magnification. The number of blood vessels in four different regions under 400 × magnification was also assessed. An area was considered microvascular when the lumen diameter was < 50 um (the equivalent of six red blood cells) and was excluded when the lumen diameter was > 50 um or vessel walls contained muscle. Stained single cells or groups of cells stained with no lumen were regarded as independent micro-vessels. The MVD was determined by counting the number of CD31-positive micro-vessels.