Chromogranin a

Food antigens [cow’s milk (Organic Valley Family of farms), and egg (Jay Robb Enterprises Inc) and ovalbumin) were conjugated to Alexa Fluor 647 dye as described by the manufacturer (ThermoFisher Scientific). The model antigens and inhibitors used were as follows: dextran, tetramethylrhodamine, 10,000 MW, lysine fixable and diamidino-2-phenylindole (DAPI) (ThermoFisher Scientific); 8-Br cADPR (Sigma); Tropicamide (SantaCruz); LY294002 (Cell signaling). The reagents used are as follows: Fluo4 AM and Imject Alum Adjuvant (ThermoFisher Scientific); RU486, polybrene, tamoxifen, carbachol, histamine and ovalbumin (Sigma Aldrich), human and mouse IL-13 (PeproTech), and mouse anti-IgE antibody, clone EM-95 (provided by Fred D. Finkelman at CCHMC). Antibodies that we used are as follows: Chromogranin A (Immunostar), MMP7 (R&D systems), mouse MCPT-1 (eBioscience), MUC2, STAT6, IL-4Rα, phospho-STAT6 (SantaCruz), phospho-AKT (Ser473) and phospho-AKT (Thr308) (#9275, Cell Signaling), actin (A2066, Sigma), DCLK1 and anti-GFP (Abcam), PerCP-Cy5.5 conjugated CD45R/B220, CD8, Ly-6G/Ly-6C, CD11c, and CD3e, PE/Cy7 conjugated ckit, biotin conjugated ST2 (BioLegend), streptavidin conjugated APC-Cy7 (BD Pharmigen), and APC conjugated FcϵR (BioLegend), Donkey anti-mouse, -rat, -rabbit & -goat conjugated to Alexa Fluor-488 or −647 (Invitrogen).

Small intestine and colon from sacrificed mice were placed immediately into 10% neutral buffered formalin (NBF). Intestines were opened longitudinally, cleaned, and swiss-rolled. Swiss rolls were fixed for ~24 hr in 10% NBF at room temperature (RT). Fixed tissues were embedded in paraffin and stained with H&E. Adenomas were graded by inspection of H&E-stained sections.
For immunohistochemistry (IHC), primary antibodies (Abs) recognized were β-catenin (Cell Signaling Technology, #2677), c-Myc (Santa Cruz, #SC764, N-262), lysozyme (#A0099), ephrinB1 (R&D Systems, clone AF473), EphB2 (R&D Systems, clone AF467), EphB3 (R&D Systems, clone AF432), cleaved caspase-3 (Cell Signaling Technology, #9661), Ki67 (Dako, Tec3 clone), chromogranin A (Immunostar, #20085), p21 (Santa Cruz Biotechnology, clone M19), GFP (Novus, 100–1678), cyclin D1 (Cell Signaling Technology, #2978), cyclin E1 (Abcam, ab7959), cyclin A1 (Santa Cruz Biotechnology, sc-751), and B23 (Sigma, B0556). Anti-LFABP antibody was the kind gift of Dr. Jeffrey Gordon (Washington University, St. Louis).

Intestinal tissue was fixed overnight in 10% neutral buffered formalin and stored in 70% ethanol until further histologic processing. Intestinal cultures were fixed in 10% buffered formalin for 5 minutes then resuspended in 30 μL preheated Histogel (American MasterTech) and stored in ethanol as above. Specimens were embedded in paraffin and sectioned at 3 μm thickness. Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) were performed per standard protocol (Lahar et al. 2011 (link); Jabaji et al. 2013 (link)). Staining was done using antibodies against CD10 (56C6, Dako, Carpinteria, CA), lysozyme (ab74666, Abcam, Cambridge, MA), chromogranin A (20086, Immunostar, Hudson, WI) (Gonzalez et al. 2013 (link)), villin (1D2C3, Dako), E-cadherin (NCH-38, Dako), β-catenin (β-Catenin-1, Dako), p120-catenin (98, Ventana, Tucson, AZ), and cytokeratin 8 and 18 (EP17/EP30, Dako). Goblet cells were identified with the periodic acid-Schiff (PAS) stain (Brown et al. 1988 (link)). Porcine ISEMFs were plated in 0.95 cm² culture wells, fixed as above, and stained with antibodies against α-smooth muscle actin (α-SMA, M0851, Dako), desmin (M0760, Dako), and vimentin (ab92547, Abcam). For immunofluorescent stains, conjugated goat secondary antibody (Invitrogen) was added at 1:200 dilution and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen).