Tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Histological and immunohistochemical staining was performed using standard techniques as described previously. 27 In brief, the embedded tumor tissues were sectioned and the slices were blocked with 10% normal goat serum (Beijing four Zheng Bo Biological Technology Co., Ltd, Beijing, China) and incubated with primary antibody for 3 h at 37°C. Subsequently, the slices were incubated with horseradish peroxidase (HRP)conjugated anti-rabbit IgG (Beijing four Zheng Bo Biological Technology Co., Ltd) for 1 h under room temperature. After washing with PBS, immunoreactivity was visualized with diaminobenzidine (DAB; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd, Beijing, China) as chromogen under a light microscope. Rabbit anti-GFP antibody (1:500; AnaSpec, Fremont, CA, USA) and rabbit anti-laminin (1:1000; Sigma-Aldrich, Milwaukee, WI, USA) were used as primary antibodies. Hematoxylin/eosin (H&E) staining was performed after tissues were fixed with 4% paraformaldehyde and embedded in paraffin.
The intratumoral microvessel density (MVD) was determined on H&E-or laminin-stained tumor tissues. Five fields were selected randomly from each tumor tissue section and quantitative analysis of the positively stained density was performed using Optimas image analyzer (Optimas Corporation USA).
The intratumoral microvessel density (MVD) was determined on H&E-or laminin-stained tumor tissues. Five fields were selected randomly from each tumor tissue section and quantitative analysis of the positively stained density was performed using Optimas image analyzer (Optimas Corporation USA).