Ki67 cell proliferation kit

For cultured cells, Ki67 staining was carried out using a Ki67 Cell Proliferation Kit (Sangon Biotech, China) according to the manufacturer's recommendations. In brief, cells were rinsed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 10 min. After blocking with 10% FBS in PBS for 1 h, cells were incubated with a rabbit anti-Ki67 antibody (1:100, Sangon Biotech) overnight at 4°C followed by an incubation with a Cy3-conjugated goat anti-rabbit IgG (red, 1:100, Sangon Biotech) for 1 h. Nuclear were counterstained with DAPI (blue).
For tumors from mice model, Ki67 were detected by standard immunohistochemistry protocols. Slides were deparaffinized, hydrated and boiled in EDTA buffer (pH 9.0) for 2 min for antigen retrieval. After treated with 3% H₂O₂ for 25 min, slides were blocked with 3% BSA for 30 min, and then incubated with a rabbit anti-Ki67 antibody (1:100, GB13030-2, Servicebio, China) overnight at 4°C, followed by an incubation with HRP-conjugated secondary antibody (Servicebio) for 50 min. Then, the signal was developed by in DAB (brown) solution for 5 min and the nuclear were counterstained with hematoxylin (blue).

Ki67 Cell Proliferation Kit was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The experiment was performed based on their protocol. The section of gastric cancer tissues were dewaxed, hydrated, and washed twice with PBS for 5 min. After blocking with Blocking Buffer for 30-60 min at room temperature, Anti-ki67 Rabbit antibody (1:50) was added to incubate overnight at 4 °C in a humidified atmosphere. Then, secondary antibody HRP-conjugated Donkey anti-Rabbit IgG (1:500) was added and incubated for 60 min at room temperature. After washing 3 times with PBS, DAB mixture was added to each slide, and incubated for 5-10 min at room temperature protected from light. The section was washed, counterstained, dehydrated, transparentized and mounted. Images were captured using microscope.