To assess the effects of DSS on mucus permeability the selleck chem apical solution was replaced by KREB solution containing either 3% DSS or 3% Dextran as described above. A second XY stack was taken after 15 min of incubation in DSS or Dextran. Images were processed using the Laser sharp 2000 software and Image J. The Z-axis section was used to present the results. DSS treatment of mice 3% DSS or FITC-DSS was administered orally in the drinking water for 12 to 120 h starting at 8.00 p.m. (dark) to assure activity of the animals at the start of the experiment. The DSS had a molecular mass of 49 kDa and had 17% sulfate substitution. The FITC-labeled DSS had 1.6 mg FITC/g DSS. Each experimental time point included 3 animals except for the 120 h control where one animal was used.

In vivo mucus measurements after DSS treatment In vivo measurements of the firm mucus were performed as described previously on animals subjected to 3% DSS in the drinking water for 24 h (n=3) or controls (n=6) [2], [30]. During the 1 h stabilization period spontaneous mucus secretion occurs producing the full mucus layers in the measurement chamber. The secreted mucus is not subjected to additional DSS. Preparation and extraction of mucus Loose and firm mucus from the in vivo measurements was collected by suctioning (loose) or gentle scraping (firm) in PBS supplemented with Complete EDTA-free protease inhibitor (Roche, Basel, Switzerland) and the samples were frozen. Total mucus from the distal colon of DSS treated animals was removed by gentle scraping in PBS supplemented with Complete EDTA-free protease inhibitor (Roche, Basel, Switzerland).

The mucus samples were extracted three times in guanidinium chloride [6.0 M GuHCl, 5 mM EDTA, 0.1 M Tris-HCl, pH 8.0] by rotation at +4��C over night and centrifuged for 20 min at 16,000��g. The resulting soluble and insoluble fractions were separated and dialyzed against water. The soluble fraction contained the luminal content including the DSS. All samples were incubated with sample buffer [0.75 M Tris-HCl pH 8.0, 2% SDS, 0.01% Bromophenol blue, 60% glycerol, 100 mM DTT] at 95��C for 10 min with continued reduction at 37��C for 2 h. SDS-agarose composite gel electrophoresis for separation of mucins The reduced samples were analyzed by composite agarose-polyacrylamide gel electrophoresis (AgPAGE) with a gel containing agarose (0.

5�C1% gradient), acrylamide (0�C6%) and glycerol (0�C10%) [31]. The electrophoresis was performed on ice at +4��C for 16 h at 12 mA/gel. The gel was stained with Alcian blue visualizing both glycosylated mucins and DSS. Histology and Immunostaining Segments of the Drug_discovery distal colon from mice were fixed in water-free Methanol-Carnoy’s fixative [60% dry methanol, 30% chloroform and 10% acetic acid]. The tissue was washed in methanol before embedded in paraffin and sectioned, 4 ��m. The sections were dewaxed using Xylene substitute (Sigma, St. Louis, MO) and hydrated.