Fig. 5.

The effect of CLCA1 on the mucus structure. (a) The effect of different classes of protease inhibitors on WT (blue) and Clca1^−/− (red) mucus growth rate. EDTA 10 mM, cOmpl 1×, E64-c 20 μM (n = 3–5). * denotes significance to Ctrl within each genotype, or as indicated by brackets. (b) Flushed distal colon explants from WT and Clca1^−/− mice were stained with fluorescent UEA1 lectin (red) to visualize the mucus. Images were acquired by confocal microscopy in z-stacks represented by x/y-axis (upper panels) and x/z-axis (lower panels). White arrows indicate axis direction; dashed lines indicate inner mucus (IM). (c) Magnified view of the mucus surface from ex vivo UEA1 lectin (grey) stained colonic mucus from WT and Clca1^−/− mice. (d) Mucus on flushed tissue stained with UEA1 were used to map isosurfaces as a measure of clumped mucus. (e) Detection of clumped mucus was quantified by mapping isosurfaces to a threshold level of UEA1 fluorescence. (f) Images of unflushed explants stained with UEA1 to detect mucus and Syto-9 to detect mucosal bacteria on WT and Clca1^−/− mouse colon explants. White lines indicate tissue surface; white arrows indicate the interface between the inner (IM) and outer (OM) mucus layers. Right panels show magnified images of mucosal bacteria (yellow boxes) and the IM/OM interface (blue boxes). Data is presented as mean ± SEM of 3–5 (a) or 4 per group (b-f) animals; ns = non-significant, *p ≤ .05, ****p ≤ .0001 as determined with two-way ANOVA followed by Tukey's MCT (A) or by Mann-Whitney test (e). All figure scale bars are 50 μm.