Bacteria and cells were fixed in 3.7% formaldehyde (v/v) in PBS for 10 min at room temperature or in methanol/acetone (1:1) for 4 min on ice for mucin staining. Samples were blocked and permeabilized (if required) in 0.5% bovine serum albumin (w/v) and 0.1% Triton X-100 (v/v) in PBS for 20 min. Specimens were incubated in primary antibodies for 60 min at room temperature. The following antibodies were used in this study: goat anti-E. coli (1:400; ab13627, Abcam), mouse anti-E. coli LPS (1:200; ab35654, Abcam), rabbit anti-R. gnavus NanH (1:200) (Owen et al., 2017 (link)), rabbit anti-L. reuteri CmbA (1:250) (Etzold et al., 2014 (link)), rabbit anti-MUC2 (1:250; sc-15334, Santa Cruz Biotechnology), mouse anti-MUC2 (1:250; sc-7314, Santa Cruz Biotechnology) and rabbit anti-occludin (1:20; 40-4700, Invitrogen). For detection, samples were incubated in donkey anti-rabbit, donkey anti-mouse or donkey anti-goat IgG conjugated with Alexa Fluor 488 or Alexa Fluor 568 (1:400; A10037, A10042, A11057, A21206, A21202, Invitrogen) for 30 min. Actin and nucleic acids were stained with fluorescein isothiocyanate-conjugated phalloidin and 4′,6-diamidino-2-phenylindole (DAPI), respectively. Samples were mounted in Vectashield (Vector Laboratories) and analysed using an Axioimager fluorescent light or LSM800 confocal laser-scanning microscope (Zeiss).
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