Immunohistochemistry (IHC) for Ki67 Protein Examination

The expression patterns, including localization and expression levels, were examined by using the IHC assay in mice tumor tissues. The detailed experimental procedures could be found in previous publications (30–32). Briefly, the mice tumor tissues were fixed by paraffin and embedded by wax, and were spliced into about 4-μm thick sections. Next, the IHC was performed by using a Benchmark XT automated staining machine (Ventana, USA), and the primary antibody against Ki67 (Ventana, USA) was diluted into 1:100 to incubate with the tumor tissues. After that, the sections were incubated with the secondary antibody labeled with horseradish peroxidase (HRP, Ventana, USA), and the 3, 3’ diaminobenzidine (DAB) was used to visualize the Ki-67 positive cells. A light microscope was employed to photograph the images to evaluate the expression status of Ki67 protein in mice tumor tissues, and the cells stained in yellow were regarded as Ki67-positive cells.