Mucus Penetrability Assay for Colon Tissue

The mucus penetrability assay was performed on distal colon tissue as previously described [16 (link),37 (link)]. Briefly, the colonic explants were mounted in the perfusion chamber with basolateral Kreb’s glucose solution containing 1μg/ml Calcein violet tissue stain (CellTrace Calcein Violet, Molecular Probes, Thermo Fisher) and a suspension of far red fluorescent beads (FluoSpheres Carboxylate-Modified Microspheres, 1.0 μm, red fluorescent (580/605), 2% solids, Molecular Probes, Thermo Fisher) and Kreb’s mannitol solution was added to the apical side and allowed to sediment for 5 min before it was refilled with Kreb’s mannitol solution. The distribution of the beads in the mucus was investigated by acquiring confocal images in XY stacks (320 x 320 μm) 30min after tissue mounting. These were obtained using a LSM700 Axio Examiner Z.1 confocal microscope with Plan-Apochromat x 20/1.0 DIC water objective (Zeiss) and the ZEN 2010 software (Zeiss). Average bead distance from the tissue surface was calculated by combining bead fluorescence intensity data for each z-plane above the tissue surface.