Ab97767

The tumor tissues were fixed in 10% of neutral formalin for 12 h, and then, dehydrated, transparent, and embedded, and made into paraffin sections with a thickness of 5 μm for immunohistochemical staining. After being dewaxed, rehydrated, and blocked, slides were incubated with CCNI2 antibody (1:100, ab97767, Abcam, Cambridge, MA, USA) or Ki67 antibody (1:200, Ab16667, Abcam, Cambridge, MA, USA) at 4°C overnight, then, washed with phosphate‐buffered saline (PBS) for 3 times, and incubated with horseradish peroxidase (HRP) conjugated goat anti‐rabbit IgG polyclonal antibody (1:400, ab6721, Abcam, Cambridge, MA, USA) at room temperature for 30 min. 3,3′‐diaminobenzidine (DAB) was applied to stain tissue slides at room temperature for 5 min in the dark, and hematoxylin (Baso Diagnostics Inc., Zhuhai, China) was used to counterstain for 10‐15 s. Photomicroscope (Olympus IX73) was used to observe and capture images. At least 10 representative fields (× 200 magnification) were selected to count the number of staining positive cells and determine the staining intensity.

After infected with CCNI2 shRNA, HCT 116, and RKO cells were collected and lysed by Cell Lysis Buffer (9803S, Cell Signal Technology, Danvers, MA). BCA Protein Assay Kit (23225, HyClone‐Pierce, Logan, UT, USA) was used to measure protein concentration. The total cellular proteins (20 μg) were subjected by 10% SDS‐PAGE for western blot (WB) analysis, and transferred to polyvinylidene difluoride (PVDF, IPVH00010, Millipore Life Science, Boston, MA, USA) membranes by wet transfer. Membranes were blocked by TBST with 5% skim milk at 4°C for 1 h, and then incubated in primary antibodies (CCNI2 antibody: 1:1000, ab97767, Abcam, Cambridge, MA, USA; GAPDH antibody: 1:3000, AP0063, Bioworld, MN, USA) at 4°C overnight. After that, horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG polyclonal antibody (1:3000, A0208, Beyotime Biotechnology, Shanghai, China) as the secondary antibody was used to incubate the membranes for 2 h at room temperature. ECL‐Plus^TM Western blotting system kit from Amersham (RPN2232, Chicago, IL, USA) was used for color developing, and chemiluminescence imaging system (AI600, GE Healthcare Life Sciences, USA) was employed to take photos, and Image J (National Institutes of Health, Bethesda, Maryland, USA) was used for immunoblotting density analysis.