Anti occludin mouse igg antibody

Caco-2 cells were grown for 5–7 days in an 8-well chamber slide (μ-Slide 8 well Glass bottom, Ibidi). Then, cells were stimulated with COF-SN (0.5 mg/ml) or OMVs (0.1 mg/ml) for 24 h at 37°C. After washing with PBS cells were fixed with 3% paraformaldehyde in PBS, permeabilized with 0.05% saponin (Sigma–Aldrich) and blocked using PBS containing 1% bovine serum albumin. The TJ proteins ZO-1, claudin-2 and occludin were stained using, respectively, anti-ZO-1 (5 μg/ml, Invitrogen), anti-claudin-2 (5 μg/ml, Abcam) rabbit IgG antibodies, and anti-occludin mouse IgG antibody (0.5 μg/ml, Invitrogen) for 16 h at 4°C, followed by incubation with Alexa Fluor 488-conjugated F(ab’)2 goat anti-rabbit IgG (H+L) (5 μg/ml, Invitrogen) or Alexa Fluor 488 - conjugated F(ab’)2 goat anti-mouse IgG (H+L) (5 μg/ml, Invitrogen) for 2 h at 37°C. Nuclei were labeled with DAPI (0,125 μg/ml, Sigma–Aldrich) for 20 min at room temperature.

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)].