4.11. Immunohistochemistry Analysis of Tumor Tissue

Immunohistochemical analysis for CD31, NOS2 and F4/80 was performed as previously described [53]. The primary antibodies were diluted as follows: anti-CD31 antibody (rabbit IgG anti-mouse CD31, Abcam, ab124432) was diluted 1000-fold, anti-NOS2 antibody (mouse IgG anti-mouse NOS2, Santa Cruz Biotechnology INC, sc-7271) was diluted 500-fold and for F4/80 mouse macrophage receptor the antibody (rat IgG anti-mouse F4/80, Bio-Rad, MCA497) was diluted 250-fold. We used the following scoring system to evaluate the area (%) of positive immunoreaction: 0.5: 5%–20%; 1: 20%–40%; 2: 40%–60%; 3: 60%–80%; 4: 80%–100%.