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.
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.
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