Anti ki67 antibody

Cells on slides were permeabilized with 0.3% Triton X-100 (Beyotime Institute of Biotechnology, Shanghai, China) for 15 min after being fixed with 4% paraformaldehyde for 15 min under room temperature. The cells were blocked using 5% goat serum (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 1 h at room temperature followed by incubation with anti-Ki67 antibody (cat. no. ab15580) or anti-CBP antibody (cat. no. ab2832) or anti-β-catenin (cat. no. ab32572) (all from Abcam, Cambridge, MA, USA; all 1:100) overnight at 4°C. The slides were then incubated with anti-rabbit Alexa Fluor 488 (cat. no. 111-545-003; 1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 h at room temperature. DAPI was used for nuclear counterstaining at room temperature for 1 h. The samples were observed under a fluorescence microscope (×40; IX73; Olympus Corporation, Tokyo, Japan).

The tumor tissues fixed in 4% paraformaldehyde (at room temperature for 24 h) were embedded in paraffin and then sectioned into 5-µm slices. After dewaxing, rehydration, and antigen retrieval, the tumor slices were incubated with 3% H₂O₂ at room temperature for 15 min. Following 15 min of blocking with goat serum (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature, tumor slices were incubated with a anti-Ki67 antibody (1:100 dilution; cat. no. A2094; ABclonal Biotech Co., Ltd.) overnight at 4°C. Subsequently, the sections were incubated with an HRP-conjugated secondary antibody (cat. no. 31460; Thermo Fisher Scientific, Inc.) at 37°C for 1 h. The sections were then stained with diaminobenzidine (DAB; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 5 min. Following DAB visualization, the sections were immersed into water and then re-stained using hematoxylin at room temperature for 3 min. The Ki67-positive cells were finally observed under a light microscope (BX53; Olympus Corporation).