Primary antibody against ki 67

IHC staining was performed as reported previously described 14 (link). In brief, paraffin-embedded tumor tissue sections were deparaffinized and rehydrated with graded ethanol. Endogenous peroxidase was blocked by 0.3% hydrogen peroxide in methanol. Antigen retrieval was done in 10 mM citrate buffer (pH 6.0) at 100°C for 15 minutes, followed by incubation with 10% BSA (Sangon, Shanghai, China) for 1 h at room temperature. After washing with phosphate-buffered saline (PBS) for three times, the slides were incubated with primary antibody against Ki67 (Cell Signaling Technology, #9449, USA) at 4°C overnight. The next day, slides were incubated with HRP (rabbit) second antibody and the immunoreactivity was generated by DAB substrate liquid (GeneTech, Shanghai, China). Finally, sections were counterstained by hematoxylin.

The tumor tissues from nude mice were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm-thick sections. The slides were subjected to antigen retrieval and incubated with primary antibody against Ki-67 (Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C, followed by treated with HRP-conjugated secondary antibody for 1 h at room temperature. Then, the tissue sections were stained with diaminobenzidine and haematoxylin. Images were captured by using a BX51 microscope (Olympus, Tokyo, Japan).

Tumour tissues were collected, fixed in 4% paraformaldehyde for 24 h at 4°C and embedded in paraffin. After 5 µm thick sections had been deparaffinized in xylene and hydrated in gradient ethanol, they were subjected to antigen retrieval using a high‐pressure method. Endogenous peroxidases were blocked by 3% H₂O₂ for 10 min at room temperature. Subsequently, sections were incubated with a primary antibody against Ki67 (cat. no. 12202, Cell Signaling Technology) diluted in working buffer (1:50) at 4°C overnight. The next day, all sections were hybridized with a secondary antibody labelled with streptavidin–horseradish peroxidase at 4°C for 50 min Subsequently, the sections were stained with DAB staining buffer, followed by haematoxylin for 25 s. The staining results were visualized by microscopy (Olympus BX41; Olympus Corporation). Positive cells labelled with brown–yellow granules in five random high‐magnification fields of each group were identified and counted by three independent pathologists. After counting the number of total cells (blue‐labelled nuclei), the proportion of Ki67‐positive cells was calculated. Cell number was also monitored by using Image‐J software using the double‐blind method. Means of different tumour tissues were used to generate the histogram.