Immunohistochemistry of Mouse Tumor Xenografts

Immunohistochemistry staining was performed using Biotin-Streptavidin HRP Detection Kit (Zhongshan Bio, Beijing, China), according to the manufacturer’s procedure.^{25 (link)} In brief, tumor tissues obtained from HepG2, CAFs combined with HepG2, or sh-VEGF-CAFs combined with HepG2 nude mice xenografts were formalin fixed, paraffin embedded, and then cut into 5-µm sections. After antigen retrieval with autoclaving in citric acid, and inactivating endogenous peroxidase with 3% H₂O₂, the slides were incubated with the rabbit anti-mouse CD31 monoclonal antibody (1:100 dilution; Abcam) overnight at 4°C. Second antibody conjugated with biotin was applied for 1 hour at room temperature. Then the sections were developed in 3,3-diaminobenzidine and counterstained with hematoxylin.