Histological and Immunohistochemical Analysis of Colonic Inflammation and Dysplasia

Histological analysis was performed as previously described [20 (link)]. In brief, fresh colon pieces were fixed in 4% Paraformaldehyde solution and embedded in paraffin. Five-micron sections of paraffin-embedded colons were stained with hematoxylin and eosin (H&E) and analyzed for severity of inflammation and dysplasia. For immunohistochemical analysis, standard immunohistochemical procedures were performed using the following antibodies: β-catenin (Cell Signaling Technology; 8480) and Ki67 (Abcam, ab16667). Intratumoral apoptotic cells were detected in paraffin-embedded colon samples with the In Situ Cell Death Kit (Roche, 11684817910) according to the manufacturer’s instructions. After the immunohistochemical staining, slides were read under microscope and fields were randomly selected for further analysis. Images were taken and the number of positive cells in each field per high-power field within each tumor from WT and Tipe^−/− tumor-bearing mice were counted. At least three fields for each mouse were used for quantification, with the animal number of five for each group.