(A) Mucus thickness in distal small intestine was measured as in Fig. 1A during 2 to 8 weeks of colonization. The mucus was removable after 4 weeks of colonization. (B–C) The mucus thickness was measured over 1 h in proximal (B) and distal (C) colon as in Fig. 1B during 2–8 weeks of colonization. (D) Mucus penetrability was analyzed in distal colon during colonization as in Fig. 1C. An impenetrable mucus developed after 6 weeks. (E) Representative pictures of confocal Z-stacks from the penetrability experiments with beads (0.5 μm red, 1 μm purple, 2 μm green) close to the epithelium (blue) in GF, 2 and 3-week colonized mice. Mucus separated the beads from the epithelium in Convr mice and after 8 weeks of conventionalization. The scale bar represents 100 μm. **, P=0.016, ***, P=0.002.