All mice were sacrificed by cervical dislocation under anesthesia with 2% isoflurane inhalation before the tumour volume reached 2000 mm3. The tumour grafts were removed, embedded in paraffin and sectioned into 3 μm sections. IHC was performed according to standard protocols. Antigen retrieval was achieved by heating the sections in 0.1 M Tris-HCl buffer (pH 9.0) at 98 °C for 15 min. Endogenous peroxidase was blocked with 3% H2O2 in PBS for 20 min. Subsequently, the sections were blocked with normal goat serum, followed by incubation with primary antibodies (mouse anti-His tag mAbs) at a 1:200 dilution overnight at 4 °C. The sections were stained using a polymer HRP detection system (DAKO, California, USA). The images were captured using an XSP-C204 (COIC, Chongqing, China).
For IF staining, the sections were blocked with 5% BSA for 1 h at room temperature and incubated with CD31 and αSMA antibodies (CST, Massachusetts, USA) for 2 h in the dark. Appropriate DyLight® fluorochrome-conjugated secondary antibodies (Earthox, Massachusetts, USA) were used at 1:200 dilutions for 1 h at 37 °C. After being immunolabelled, the sections were washed and stained with DAPI (Sigma, Mississippi, USA) for 10 min at room temperature. The images were visualized under a NIKON ECLIPSE C1 microscope (NIKON, Tokyo, Japan).
For IF staining, the sections were blocked with 5% BSA for 1 h at room temperature and incubated with CD31 and αSMA antibodies (CST, Massachusetts, USA) for 2 h in the dark. Appropriate DyLight® fluorochrome-conjugated secondary antibodies (Earthox, Massachusetts, USA) were used at 1:200 dilutions for 1 h at 37 °C. After being immunolabelled, the sections were washed and stained with DAPI (Sigma, Mississippi, USA) for 10 min at room temperature. The images were visualized under a NIKON ECLIPSE C1 microscope (NIKON, Tokyo, Japan).