One-centimeter colon samples were collected from a standard area of the proximal part of descending colon for gene expression and ELISA analyses. Samples for gene expression assay were immediately immersed in an RNA-later solution (Takara Bio Inc, Shiga, Japan) and stored at − 80°C until further processing. The remaining colon was fixed in 10% neutral-buffered formalin for histologic and immunohistochemical analyses. For histologic evaluation,

formalin-fixed colon and mesenteric lymph nodes (MLN) were embedded in paraffin, cut at 5 μm, and stained with hematoxylin and eosin or immunohistochemistry (IHC). Dysplastic and neoplastic lesions in the colonic mucosa (excluding polyps) were scored on a 0 to 4 ascending scale using previously described criteria [33]. Mucosal/submucosal inflammation SGI-1776 in vitro in the colon was scored in non-ulcerated areas based on the extent and PFT�� mouse severity of inflammatory cell accumulations. Loss of colonic epithelial integrity was scored on the basis of the extent

and severity of the typical DSS-induced colonic mucosal erosive and ulcerative lesions. Both parameters were scored semi-quantitatively on 0 to 4 ascending scales according to the following scheme: 0, normal; 1, mild; 2, mild to moderate; 3, moderate; 4, severe. Primary antibodies for IHC included 1) rabbit polyclonal antibodies against β-catenin, Cytoskeletal Signaling inhibitor myeloperoxidase (MPO; Thermo Fisher Scientific/Lab Vision, Fremont, CA), E-cadherin, IL-17, TGF-β1 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), cleaved caspase-3 (Cell Signaling Technology, Beverly, MA), and CD3 (Cell Marque, Rocklin, CA); 2) rabbit monoclonal antibodies against Ki-67 and c-kit (Cell Marque); 3) rat monoclonal antibodies against Foxp3 (eBioscience, Inc, San Diego, CA) and F4/80 (Serotec, Oxford, United

Kingdom); and 4) a goat polyclonal antibody against IL-16 (Santa Cruz Biotechnology, Inc). Heat-induced antigen retrieval was performed with citrate buffer, pH 6, for β-catenin, E-cadherin, MPO, and cleaved caspase-3, with EDTA buffer, pH 8, for IL-17 and Foxp-3, or with CC1 epitope retrieval solution for Ki-67, CD3, and IL-6 (Ventana Medical Systems, Inc, Tucson, AZ). TGF-β1 antigens were retrieved with protease (Cell Marque) and F4/80 antigens with trypsin (Thermo Fisher Scientific/Lab Vision). Rabbit primary antibody binding was detected with goat anti-rabbit polymer HRP (ZytoChem Plus, Berlin, Germany), whereas rat and goat primary antibody binding was detected with species-appropriate biotinylated secondary antibodies (Serotec) and streptavidin-peroxidase (Ventana Medical Systems, Inc). Color was developed with DAB substrate-chromogen system (DakoCytomation, Glostrup, Denmark), and tissues were counterstained with hematoxylin.