Mucins in Infectious Colitis Figure 1. C. rodentium induced colitis and infection load. The number of C. rodentium CFU in feces increased over time (A, * p,0.05 compared to pre-inoculation, # p,0.05 compared to day 7, Mann Whitney U test, n = 8). At day 12, a higher number of CFU was detected in the large intestine (LI) than in the small intestine (SI) (B,* p,0.05), and low numbers of CFU were found in the internal organs (C). Histological colitis scores were low to moderate, with the distal large intestine being worst affected (D). doi:10.1371/journal.pone.0003952.g001 for the detection of the different antibodies was either: 1) 10 mM citric acid, pH 6 at 95uC for 20 min (CT2, Muc17/Muc3-S2); 2) 10 mM citric acid, pH 6 at 95uC for 20 min followed by 10 mM 1,4-dithiothretiol in 0.1 M Tris/HCl buffer, pH 8 at 37uC for 30 min and then 25 mM iodoacetamine in the dark for 30 min (MM2-2); 3) High pH Antigen Retrieval Solution (Dako, RC20); or 4) Rodent decloaker (Biocare Medical, 45M1) at 80uC for 2 h. Sections were then treated with 3% (v/v) hydrogen peroxide for 10 min at room temperature. The sections were washed 3 times between all subsequent steps in phosphate buffered saline, pH 7.4 (PBS) containing 0.05% Tween-20. Non-specific binding was blocked by 10% Milk Diluent/Blocking solution (KPL, Maryland, USA) in PBS (CT2, MM2-2, Muc3-S2, hHAI-B-1 and RC20) or rodent block M (Biocare Medical, 45M1) for 30 min. The sections were incubated with a primary antibody diluted in 10% Milk Diluent/Blocking solution (KPL, Maryland, USA) in PBS containing 0.05% Tween-20 (CT2 1:50, MM2-2 5 mg/ml, Muc17/Muc3-S2 1:2000, hHAI-B-1 1:200, 45M1 1:1000 and rM13.C 30 mg/ml,) for 1 h, then incubated with anti-rabbit antibody conjugated to horseradish peroxidise (HRP), MM2-2, Figure 2. Serum Ig response to C. rodentium. Serum concentra- tions of Ig reactive with C. rodentium lysate determined by ELISA in 8 mice prior to and 1, 7 and 12 days following infection. doi:10.1371/journal.pone.0003952.g002 PLoS ONE | www.plosone.org 3 December 2008 | Volume 3 | Issue 12 | e3952