4]. The apical side of the chamber was bathed in 100 ��l oxygenated KREB solution with supplents [5.7 mM Na-Pyruvate, 5.1 mM Na-L-Glutamate and 10 mM D-Mannitol, pH 7.4]. The temperature was kept at 37��C throughout the whole experiment. Biopsies obtained from sigmoid colon were instantly put into ice-cold oxygenated KREB solution and kept on ice until mounting neither in the RC-50 imaging chamber. The following procedure was identical to the one described for mouse tissue. The thickness of the mucus layer was assessed by measuring the distance between the epithelial surface and the surface of the mucus layer using a micropipette (Sutter instruments, CA) connected to a micropuller (55�� angle) (in-house made) and observed through a stereomicroscope (Leica, Wetzlar, Germany).
Digital recording of the measurements was enabled by connecting the micropuller to a digimatic indicator (Mitutoyo, Kawasaki, Japan). To visualize the surface of the mucus layer a suspension of activated charcoal was added. The thickness was measured with 15 min intervals for a total time of 60 min. During each measuring event five recordings were made and the calculated mean value was used as a single measurement. The vertical distance between the epithelial surface and the mucus surface was calculated by multiplying the obtained value with cosin55��. At time 45 min the loose mucus layer was removed by suction and the thickness of the firmly adherent mucus layer was measured. To assess the effect of DSS and Dextran on the thickness of the mucus layer the apical solution was replaced with KREB mannitol solution containing either 3% DSS or 3% Dextran and incubated for 15 min.
After 15 min of incubation the loose mucus layer was removed by suction and the thickness of the firmly adherent layer was measured. Data is presented as mean �� SEM. Effects of the treatments were analyzed by using the student’s t-test. A p-value<0.05 was considered as statistically significant. Confocal microscopy of the effects of DSS on mucus properties The effects of DSS on mucus permeability were studied using the RC-50 imaging chamber mounted onto an upright confocal microscope. The protocol used for isolating the tissue, running the chamber and the solutions used in these experiments were the same as described for the mucus thickness measurements.
The colonic epithelium was labeled using CellTracer BODIPY GSK-3 TR methyl ester (Invitrogen, Carlsbad, CA) added to the KREB solution and the basolateral perfusate (2 ��l/ml). An additional incubation of 20 min in KREB-Bodipy solution was added after removing the muscle layer. After 45 min incubation in the RC-50 imaging chamber yellow-green fluorescent beads (2 ��m FluoSpheres, Invitogen, Carlsbad. CA) were added to the apical surface and allowed to sediment down to the mucus layer. Confocal images were taken in a XY stack with an optical section of 13.