Immunohistochemical kit

All mice were euthanized on the 27th day after administration. The tumor tissues were removed and fixed in 4% paraformaldehyde, and then the immunohistochemistry staining was performed on paraffin-embedded tissues. Briefly, the sections preparation and procedures were performed according to the protocol of the immunohistochemical kit (ZSGB-BIO, Beijing, China). Sections were subsequently incubated with the monoclonal antibodies of mouse CD31 (0.5 µg/ml, Abcam, USA), rabbit ki67 (1:150, Abcam, USA) or rabbit MMP-9 (1:200, Abcam, USA) at 4 ℃ overnight. Then the Biological and Pharmaceutical Bulletin Advance Publication sections were covered using the HRP conjugated from the corresponding species of primary antibody for 1 h at room temperature. Next, the sections were incubated with diaminobenzidine (DAB) and counterstained with hematoxylin. For apoptosis analysis, the sections were stained using the TdT-mediated FITC-dUTP nick end labeling (TUNEL) and counterstained with DAPI according to the manufacturer's protocol (in situ cell death detection kit, Roche, UK). The images were taken by the positive fluorescence microscope (Ni-U, Nikon, Japan), and the positive cells from three random areas of each section were measured by determining the integral optical density (IOD)/Area using the Image-Pro Plus 6.0 software (Media Cybernetics, MD, USA).