Goat anti e coli

Samples were fixed in 3.7% formaldehyde in PBS for 20 min and blocked/permeabilized with 0.1% Triton X-100 and 0.5% bovine serum albumin in PBS for 20 min. Samples were subsequently incubated in primary antibodies (goat anti-E. coli from Abcam; rabbit anti-EspA from Gad Frankel, Imperial College London; mouse anti-Tir from John Leong, Tufts University, USA) for 60 min, washed, and incubated in Alexa Fluor-conjugated secondary antibodies (Invitrogen) for 30 min. Filamentous actin was labeled with fluorescein isothiocyanate-conjugated phalloidin (Sigma). Samples were mounted in Vectashield medium (Vector Laboratories) and analyzed using a fluorescence light microscope (Axiovert 200M; Zeiss).

Biopsy samples with preserved mucus layer were embedded in OCT compound (Sakura), snap-frozen in a dry ice/ethanol bath and stored at –70°C until use. Serial sections of 7 μm were cut with a Microm HM550 cryostat (Thermo Scientific), picked up on poly L-lysine-coated slides and air-dried. Tissue sections were blocked with 0.5% bovine serum albumin (BSA) in PBS for 20 min. Cells on coverslips were fixed in 3.7% formaldehyde in PBS for 10 min or in methanol/acetone (1:1) for 4 min on ice (for mucus staining) and blocked/permeabilized with 0.1% Tx-100 and 0.5% BSA for 20 min. Cells and cryosections were incubated with primary antibodies (rabbit anti-CmbA (Etzold et al., 2014b (link)), rabbit anti-MUB (MacKenzie et al., 2010 (link)), rabbit anti-SRR (kind gift from Donald MacKenzie), goat anti-E. coli from abcam, mouse anti-MUC2 from Santa Cruz) for 60 min, washed and incubated with Alexa Fluor-conjugated secondary antibodies (Life Technologies) for 30 min. Cell nuclei and filamentous actin were stained with DAPI (Roche) and FITC-conjugated phalloidin (Sigma), respectively. Samples were mounted in Vectashield (Vector Laboratories) and analyzed using a fluorescence light microscope (Axiovert 200M, Zeiss). Formation of EPEC actin pedestals or microcolonies was quantified from ten random fields of view containing around 70 cells for each experimental condition.

T84 cells on filters were fixed in 3.7% formaldehyde in PBS for 10 min and blocked/permeabilized with 0.1% Triton X-100 (Tx-100) and 0.5% BSA in PBS for 20 min. For occludin staining, cells were pre-extracted as recommended by the manufacturer. Cells were subsequently incubated in primary antibodies (polyclonal goat anti-E. coli from abcam, polyclonal rabbit anti-occludin from Invitrogen) for 60 min, washed and incubated in Alexa Fluor-conjugated anti-goat or anti-rabbit IgG (Invitrogen) for 30 min. Cell nuclei/bacterial DNA and filamentous actin were labelled with DAPI (Roche) and FITC-conjugated phalloidin (Sigma) respectively. Filters were mounted in Vectashield (Vector Laboratories) and analysed using a fluorescence light microscope (Axio Imager, Zeiss) or confocal laser scanning microscope (LSM 510 Meta, Zeiss).