Fluorescence In Situ Hybridization.

FISH was performed as described previously (65). Briefly, distal colons from adult male mice (aged >12 wk) were fixed in Carnoy’s fixative overnight. Paraffin-embedded sections were dewaxed, hydrated, and incubated with 1 μg of Alexa 488-conjugated EUB338 (5′-GCT​GCC​TCC​CGT​AGG​AGT-3′; Integrated DNA Technologies) in 200 μL of hybridization buffer (750 mM NaCl, 100 mM Tris⋅HCl, pH 7.4, 5 mM EDTA, 0.01% BSA, 10% dextran sulfate) at 40 °C for 16 h. Sections were rinsed with wash buffer (50 mM NaCl, 4 mM Tris⋅HCl, pH 7.4, 0.02 mM EDTA) briefly, and then washed at 45 °C for 20 min. After washing with PBS three times for 5 min, samples were incubated with UEA-1 (DyLight 594; 1:100; Vector Laboratories) for 1 h at 4 °C. After three more PBS washes for 5 min, samples were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories). The distance between the epithelial surface and luminal bacteria was measured at four points of each colon section per mouse by ImageJ (NIH). The average of four points is shown as a dot.