Genomic DNA from tail biopsies was digested with EcoR1 overnight and 10 μg of digested DNA was resolved in 1% agarose by electrophoresis. Serial dilutions of plasmid containing the CD68TGF-βDNRII were included as a positive control. Gels were denatured, neutralized, and cross-linked using standard protocols. 32P-labeled probe was used for hybridization (49°C) and visualization via autoradiography. DSS (41 kDa) (ICN Biomedical) was used to supplement the drinking

water of study animals for 6 days as 1.5, 2, or 2.5% (w/v) solution. Fresh solution was replaced at day 3. After day 6, mice were returned to normal water and monitored for an additional 8 days. Body weight, appearance, occult blood in feces Hem occult test (Beckman Coulter), stool consistency, and diarrhea were

recorded daily from coded animals. Dabrafenib supplier At time of sacrifice, mice were evaluated for colon length. Disease activity index (DAI) was derived through the evaluation of appearance/activity, diarrhea, and rectal bleeding. DAI=(appearance/activity)+(diarrhea score)+(rectal bleeding score). DAI has a maximum score of 5 determined as follows: Appearance/activity score (0, normal grooming and active versus 1, lack of grooming and lacking normal activity), diarrhea score (0, solid formed stool; 1, loose formed stool; and 2, watery fecal Torin 1 cost matter), rectal bleeding score (0, no blood; Carbohydrate 1, positive hem occult test; 2, gross bleeding from rectum). Approximately, 1 length of distal colon was removed, fixed in 10% buffered formalin overnight, and kept in 70% ETOH until processing. Tissue was embedded

in paraffin and for each colon sample 5 μm sections were cut and stained with H&E or Periodic acid-Schiff (PAS) and examined by light microscopy. Colonic inflammation was evaluated in a blind manner by two observers that estimated the following: (i) percentage of involved area, (ii) amount of follicles, (iii) edema, (iv) erosion/ulceration, (v) crypt loss, (vi) infiltration of polymorphonuclear cells, and (vii) infiltration of mononuclear cells. The percentage of area involved, erosion/ulceration, and the crypt loss was scored on a scale ranging from 0 to 4 as follows: 0, normal; 1, <10%; 2, 10–25%; 3, 25–50%; and 4, >50%. Follicle aggregates were counted and scored as follows: 0, zero to one follicle; 1, two to three follicles; 2, four to five follicles; and 3, six follicles or more. The severity of the other parameters was scored on a scale from 0 to 3 as follows: 0, absent; 1, weak; 2, moderate; and 3, severe. All scores on the individual parameters together could result in a total score ranging from 0 to 24 47. Peritoneal Mϕs were harvested on day 4 following administration of 4% thioglycollate (Fisher scientific).