Mucins in Infectious Colitis Figure 3. Tissue localization of mucins in small intestine and caecum. Histologically, the small intestinal villi and caecal crypts are covered by a simple columnar epithelium consisting of enterocytes with interspersed goblet cells. Using immunohistochemistry, we demonstrated that the apical surface of small intestine and caecal epithelia are lined by the cell surface mucins Muc13 and Muc17 as well as a small amount of Muc4 (brown). The goblet cells contain alcian blue positive material (blue), which mainly is Muc2 (brown), but also some Muc4. The photographs were taken using a 206 magnification. The quantification scores for any of these mucins were not significantly different between non-infected mice (#) and mice infected with C. rodentium for 12 days (N) in these areas of the intestine. (Mann Whitney U test, n = 7). SCL = subcellular localization, RC = region of crypt. The mucin stains are shown in serial sections on the same tissue area, whereas the mucin stain scores represent the average stain scores for the entire specimen for each individual mouse. doi:10.1371/journal.pone.0003952.g003 RC20) or anti-hamster antibody conjugated to biotin followed by streptavidin-HRP (CT2) for 1 h or with MM HRP-Polymer (Biocare Medical, 45M1) for 20 min. Bound antibody was visualized with diaminobenzidine for 10 min. The sections were counterstained with Harris’s haematoxylin. Gastric sections were used as positive controls for CT2 and 45M1. Human samples: Formalin-fixed, paraffin-embedded tissue sections (4 mm) of archived biopsies were dewaxed and rehydrated. Sections were treated with 1% periodic acid to expose the epitope and then with 3% (v/v) hydrogen peroxide for 30 min at room temperature. The sections were washed 3 times between all subsequent steps in 0.15 M NaCl, 0.1 M Tris/HCl buffer (pH 7.4) containing 0.05% Tween-20. Non-specific binding was blocked by protein block (Dako) for 30 min. The sections were incubated with the BC2 antibody diluted in Antibody Diluent for 1 h, then incubated with Broad Spectrum Poly HRP Conjugate (Zymed Laboratories inc, San Fransisco, USA) for 10 min and then with diaminobenzidine for 10 min. The sections were counterstained with Harris’s haematoxylin. Staining was classified into one of four categories: high level of staining (approx. 90–100% of cells staining, score = 3), medium level of staining (25–90%, score = 2), low level of staining (1–25%, score = 1) and virtually no staining (,1%, score = 0). The scoring of the stain was performed by an individual blinded to the infection status of the mice. Two of the stains were also analyzed by a second individual blinded to the infection status of the animals, and the results between the two scorers were consistent. PAS/Alcian blue stain De-waxed sections were immersed in 100% ethanol for 10 min, rinsed in water for 10 min, immersed in 3% acetic acid for 2 min and stained in 1% Alcian Blue 8GX in 3% acetic acid (pH 2.5) for 2.5 h. Non specific stain was removed with 3% acetic acid and rinsed in water for 10 min. The slides were then oxidized in 1% periodic acid in water at room temperature for 10 min, washed in water for 5 min, immersed in Schiff’s reagent for 10 min, rinsed in water for 5 min and then three times in 0.5% sodium meta- bisulphite before a final wash in water. To reveal O-acetylated oligosaccharides sections were first treated with 0.1 M KOH for 30 min and then 1 mM periodic acid prior to the Schiff reagent. Antibody response against C. rodentium C. rodentium were harvested into PBS, washed once and then sonicated 5 times while on ice with a 3 min interval between sonications. After sonication, the solution was centrifuged at 4000 g for 4 min. The supernatant was diluted to A280 = 0.4 and plated onto Maxisorb plates (Nunc). The microtiter plates were washed 3 times in PBS pH 7.4 containing 0.05% Tween-20 between all ensuing steps. Unbound sites were blocked with 1% bovine serum albumin (BSA) for 1 h. The microtiter plates were incubated with murine sera diluted 1:100 in PBS containing 1% BSA and 0.05% Tween, pH 7.4, for 1 h at room temperature. The microtiter plates were incubated with HRP conjugated anti mouse Ig diluted 1:2000. Bound secondary antibody was visualized using BD OptEIA TMB substrate reagent (BD, San Diego, USA). The reaction was stopped with 1 M sulphuric acid and absorbance at 450 nm was measured. C. rodentium binding to Muc2 The insoluble complex of Muc2 was extracted from murine intestinal mucosal scrapings as previously described [42]: mucosal scrapings were gently dispersed with a Dounce homogenizer in 6 M guanidinium chloride, 5 mM EDTA, 5 mM N-ethylmalei- mide, 10 mM sodium phosphate buffer, pH 7.0, and left stirring at 4uC overnight. After centrifugation (21,000 g, 4uC, 60 min), the soluble material was removed, and the pellets were re-extracted twice, as described above. The final extraction residues (insoluble glycoprotein complex) were solubilized by reduction in 6 M guanidinium chloride, 10 mM dithiothreitol, 5 mM EDTA, 0.1 M Tris-HCl buffer, pH 8.0, at 37uC overnight and then alkylated with 25 mM iodoacetamide in the same buffer and then dialyzed against 4 M guanidinium chloride. After centrifugation, the supernatant was retained. This solubilised ‘‘insoluble’’ MUC2 complex was diluted in 4 M guanidinium chloride and 100 ml/ well were coated onto microtiter plates (NUNC F96; Polysorp, Roskilde, Denmark) overnight at 4uC. The microtiter plates were washed 3 times in PBS pH 7.4 containing 0.05% Tween-20 between all ensuing steps. Unbound sites were blocked with 0.5% bovine serum albumin in 1% blocking reagent for ELISA in PBS containing 0.05% Tween-20 for 1 h. The microtiter plates were incubated with biotinylated C. rodentium in 1% in blocking reagent for ELISA containing 0.05% Tween pH 7.4 for 1 h at room temperature. The microtiter plates were incubated with HRP conjugated streptavidin diluted 1:2000. Bound secondary antibody was visualized using 2,29-azinobis(3-ethylbenzothiazoline)-6-sul- phonic acid as a substrate in citric acid- Na2HPO4 buffer, pH 4.3. Absorbance at 405 nm was measured after 40 min. When the 6 M guanidinium HCl-insoluble fraction containing Muc2 was sub- jected to agarose gel electrophoresis and then blotted onto a membrane, the only PAS positive band present corresponded exactly to the MU2 band identified by Western blotting. However, we have not performed Western blotting for other mucins and very small amounts of other contaminating mucins cannot be excluded. Statistics CFU data were log transformed. Individual data points are presented and the non-parametric Mann Whitney U test was applied to ascertain differences. All statistics were determined using Systat 12 (Systat Software, Inc., San Jose, USA). Results C. rodentium infection and associated pathology is greatest in the large intestine Before inoculation, C. rodentium was not detected in the murine feces (Figure 1A). On day 7, C. rodentium was detected in feces of 5/ 8 mice and the mean C. rodentium content in feces was 103 CFU/g. By day 12, C. rodentium was detected in feces of 6/8 mice and the mean C. rodentium content in feces had increased to 105 CFU/g PLoS ONE | www.plosone.org 5 December 2008 | Volume 3 | Issue 12 | e3952