Immunohistochemistry (IHC) for Ki67

IHC was performed according to the manufacturer's instructions of KI67 Cell Proliferation kit (Sangon Biotech Co., Ltd.) to detect Ki67 expression in the transplanted tumor. Briefly, fresh tumor tissue samples from the negative control (PBS), low-dose treatment (25 µmol/l 17BIPHE2) and high-dose treatment groups (35 µmol/l 17BIPHE2) were fixed by soaking in 10% formalin and were embedded in paraffin overnight at 65°C. Then, the samples were sectioned at 4-µm thickness after heating for 10 min at 37°C, deparaffinized in xylene, and rehydrated in graded ethanol [100 (twice), 90, 80, 70, 50 and 0%, each for 5 min). The tissue sections were immersed into a dye vat filled with the antigen repair solution and incubated under high pressure for 5 min in a pressure cooker to remove the endogenous peroxidase. After removing the water from the tissue sections, the samples were incubated with 3% H₂O₂ in a wet box for 15 min at room temperature and washed thrice with PBS for 5 min each to remove the excess H₂O₂. The slides were then incubated with goat serum (Gibco; Thermo Fisher Scientific, Inc.) for 20 min at room temperature. Subsequently, the slides were incubated overnight with mouse anti-Ki67 (cat. no. PB9026; 1:200; Wuhan Boster Biological Technology, Ltd.) antibody at 4°C. The next day, the samples were removed and equilibrated at room temperature for 30 min. The primary antibody was washed off with PBS. Next, the slides were incubated with reagent A at 37°C for 20 min, washed thrice with PBS for 5 min each, and incubated with reagent B at 37°C for 30 min. The slides were washed thrice with PBS for 5 min each and then incubated with DAB (1:20) for 2–10 min for color development, and the reaction was stopped using pure water. Then the nuclei were re-stained for 4 min with hematoxylin at room temperature, rinsed thoroughly with pure water and dipped in tap water until the color returned to blue. All slides were dehydrated with graded ethanol [50, 70, 80, 90, 100 (twice) and 50% each for 2 min] and covered with neutral balsam for analysis under light microscope with five fields of each group.