Anti mouse ki67

Mouse blood was collected from retinal venous plexus, centrifuged to harvest serum, which were stored at − 80 °C. Mice were sacrificed by cervical dislocation. The whole intestine was removed immediately after sacrifice and opened longitudinally after washed with ice-cold PBS as previously described [37 (link)]. The number, location, and size of visible tumors throughout the intestine were measured to calculate the incidence of adenoma. Tumor numbers were counted and grouped based on sizes: < 2 mm, 2–4 mm and > 4 mm. Tissue sections were fixed in 10% formalin followed by paraffin embedding. Then they were stained with hematoxylin and eosin for pathological evaluation by a pathologist blinded to the experimental groups. Histological analysis for polyp, adenoma, and adenocarcinoma was performed by a board-certified pathologist (PV) as previously described [38 ]. The histology scoring criteria is as follows: 0 = normal, 1 = moderate, 2 = marked and 3 = severe.
For the murine samples, immunohistochemistry was performed to detect total Ki67 (anti-mouse Ki67, Abcam), PCNA (anti-mouse PCNA, Abcam) and BrdU (anti-BrdU kit, Invitrogen); all stains used horseradish peroxidase-conjugated antibody, with chromogenic detection with the substrate 3–3′-diaminobenzidine, and finally counterstained with hematoxylin.

Mouse implanted tumors were dissected out and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 6 hours, and then cyro-protected in 30% sucrose for 24 hours. Frozen samples were then sectioned in 6 μm. Primary antibodies used in immunohistochemistry are rat polyclonal anti-mouse Ki-67 (1:300) (Abcam, Cambridge, MA, USA).

Immunohistochemistry (IHC) was performed in the paraformaldehyde‐fixed and paraffin‐embedded sections of mouse breast tumor tissues and lung tissues. Primary antibodies were used as follows: anti‐mouse‐Ki67 (Abcam, Cambridge, UK, ab15580); anti‐mouse cleaved Caspase 3 (Abcam, ab2302); anti‐mouse CD31 (Maixin, Shanghai, China); anti‐SENP3 (CST, Boston, CA, USA, 5591); anti‐CD206 (Abcam, ab64693). The secondary antibodies (1 : 200) were then incubated for 2 h. Sections were developed with Vectastain ABC kit (CK‐4000) and DAB (SK‐4100) detection system (Vector Laboratories, Burlingame, CA, USA) and counterstained with hematoxylin. The images were photographed, and the average optical density of at least three fields of each image was quantified and statistically analyzed by imagej software (National Institutes of Health, Bethesda, MD, USA).