Quantifying Bacterial Penetration of Gut Mucus

Bacterial penetration of the inner mucus layer was scored using the fluorescence-in situ-hybridization (FISH)-, anti-Muc2- and DNA-stained sections as described previously (S2 Table) [28 (link)]. The mucus layering score, focusing solely on the layering and structure of the inner, stratified mucus layer and the goblet cell filling score [16 (link)] were applied (S3 Table and S4 Table, respectively). Sections were scored independently by two blinded individuals, except for the naive controls which were scored by one blinded individual using the Eclipse E1000 and Eclipse90i (Nikon) fluorescence microscope as well as the Axiovert 200M microscope (Zeiss) with the EXFO X-Cite 120 Fluorescence Illumination System (Olympus, Hamburg, Germany). Micrographs were obtained using the LSM700 Axio Examiner Z.1 confocal microscope (Zeiss) with the ZEN 2010 software (Zeiss).