In vivo analysis of micro-vessel density (MVD)

To quantify tumor angiogenesis, MVD was quantified. First, micro-vessels were immunostained with anti-CD31 antibody as follows: Tumor frozen sections were sliced to 6-μm thickness, dried for a few hours at room temperature, and fixed in 10% formalin. Sections were washed with PBS thrice and the endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 20 min at room temperature. Sections were then blocked for 30 min with 2% normal swine serum (VECTOR Laboratories, Brussels, Belgium) in PBS. Incubation with primary polyclonal rabbit anti-CD31 antibody (1:50) was performed at 4°C overnight. Slides were then incubated with a biotinylated secondary antibody (VECTOR Laboratories) (1:200) for 30 min at room temperature. Slides were incubated with VECTASTAIN Elite ABC Kit (VECTOR Laboratories). 3,3′-Diaminobenzidine (DAB) staining was performed (DAB TRIS Tablet, Muto Pure Chemicals, Tokyo, Japan) to detect CD31 and the nuclei were counterstained with Mayer's hematoxylin (Muto Pure Chemicals). MVD was then analyzed as follows: Slides were scanned under low power field (×40) to select micro-vessels in the densest areas. CD31 immunopositive pixels per microscopic field were counted under high power objective (×200) using Image J software (NIH, Bethesda, MD, USA). MVD was quantified as the percentage of CD31 immunopositive pixels per high-power field in 10 views.