Hemoccult test kit

DSS (Molecular weight 36,000-50,000 kDa) was obtained from MP Biomedicals (catalog no. 02160110). Hemoccult Test Kit was obtained from Beckman Coulter. Complete EDTA-free protease inhibitor cocktail was from Roche.
Primary antibodies used for immunohistochemistry (IHC) and western blotting (WB) were: mouse anti-actin (Millipore MAB1501R, diluted 1:1000 for WB), mouse anti-β-catenin (BD Transduction Laboratories #610154, diluted 1:1000 for IHC and 1:2000 for WB), rabbit anti-Ki67 (Abcam Ab15580, diluted 1:500 for IHC), rabbit anti-EGFR phosphorylated at Tyr1068 (Abcam Ab40815, diluted 1:200 for IHC). Rabbit anti-PACS-2 18193 was previously described [21 (link)], and was diluted 1:1000 for WB.
Enzyme-linked immunosorbent assay (ELISA) kits used for analysis of ex vivo colonic tissue culture supernatants were mouse amphiregulin DuoSet ELISA (R&D Systems DY989) and mouse TNF-α ELISA Ready-SET Go! (eBioscience 88-7324).
The primers used for Pacs2 genotyping were 5′-ATG CAT ACC TGC CCT TAG CAG AGG-3′, 5′-TGG AGT CTG AGG TTG AGG CCT TGA G-3′, and 5′-ATG GCG TTA CTT AAG CTA GCT TGC-3′. Primers for Apc^Min/+ genotyping were 5′-GCC ATC CCT TCA CGTTAG-3′, 5′-TTC CAC TTT GGC ATA AGG C-3′ and 5′-TTC TGA GAA AGA CAG AAG TTA-3′. All primers were obtained from TAG Copenhagen.

The disease activity index (DAI) assessment was used to assess colitis severity. It is a compilation of multiple assessments, including the percentage of body weight loss, stool consistency, and fecal blood. A Hemoccult test kit (Beckman Coulter Inc., Brea, CA, USA) was used to determine occult blood in the stool samples according to manufacturer instructions. DAI assessment was conducted in a blinded fashion, starting 1 day prior to DSS treatment (day 0), occurring throughout the second cycle of 2% DSS administration, and continuing until 2 days after DSS exposure, meaning that the assessment ran for a total of 5 days, as previously described [127 (link),128 (link),129 (link),130 (link),131 (link)]. Each measurement was scored on a scale ranging from 0 to 4, which was then summed for a DAI score per mouse. A maximum severity score of 12 was possible. To normalize the findings, the daily DAI score per mouse was subtracted from its respective day 0 score. On the 8th week, the animals were euthanized, and their brains and colons were collected for further analysis.

Colitis was induced in mice by oral ingestion of 3% DSS (SKU 02160110-CF; MP Biomedicals) in drinking water for 5 days. The severity of colitis activity was graded on designated dates as described (33 (link)). Body weight, occult or gross rectal bleeding, and feces consistency (on scales of 0 to 4) were monitored for each mouse. The resultant inflammatory bowel disease (IBD) disease activity index is the average of the scores of the colitis symptoms. The occult blood in mouse fecal samples was detected using Hemoccult Test Kit (Beckman Coulter Inc., Fullerton, CA).