Caco-2 cells were grown in an 8-well chamber slide (Ibidi) for 6 days. After EPEC infection in the absence or presence of COF-SN or OMVs, cells were washed, fixed and permeabilized as described before [31 (link)]. Nuclei were labelled with DAPI (0.125 μg/ml), and occludin and ZO-1 were stained respectively with anti-occludin mouse IgG antibody (0.5 μg/ml, Invitrogen) and anti-ZO-1 rabbit IgG antibody (5 μg/ml, Invitrogen) [31 (link)]. F-actin was stained with phalloidin–tetramethylrhodamine B isothiocyanate (−TRITC) (dilution 1:50) as described previously [56 (link)]. Immunofluorescence was analysed in a Leica TCS SP2 confocal microscope with a 63 × 1.32NA oil immersion objective and Ar, Ar-UV and HeNe lasers. Fluorescence was recorded at 405 nm (blue; DAPI) and at 488 nm (green, Alexa Fluor 488). Images (12-bit) were obtained at a resolution of 0.232 × 0.232 × 0.488 μm/voxel (x, y, z respectively) and analysed using Fiji processing package [57 (link)].
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