Eclipse e 80i microscopy

Reactive oxygen species production was measured using DCFH-DA spectrofluorometry. After stimulation, DCFH-DA (20 µM) was added for 30 min at 37 °C in the dark. Intracellular ROS production was measured in a fluorometer (SFM 25; Kontron Instruments, Japan). For DCF fluorescence imaging, Caco-2 cells were grown on the cover glass for 3 days, fixed and permeabilized with paraformaldehyde 4% and Triton 0.2% for 30 min at 4 °C. Cells were then incubated with DCF-HA 20 µM for 30 min at 37 °C in the dark. Fluorescence images from multiple fields were obtained using a Nikon Eclipse e 80i microscopy. The images were analysed using the NiS Elements D imaging software (Nikon Instruments, Inc., NY, USA).

Actin staining: fixation and permeabilization was performed with 4% paraformaldehyde and 0.2% Triton X-100 for 30 min at + 4°C. Cells were treated with 50 µg/ml solution of Alexa Fluor 594-phalloidin (Sigma-Aldrich) for 40 min.
Occludin staining: cells were fixed by adding 100% methanol for 10 min at room temperature and probed with anti-occludin antibody (Abcam ab59720) over night at +4°C. Bound antibody was detected with Alexa fluor 488 conjugated anti-rabbit IgG antibody (Invitrogen, A21206).
Immunofluorescence assay for NF-kB: Caco-2 cells were grown on the chambered cover glass for 3 days, fixed with 4% buffered paraformaldehyde (pH 7.4) for 30 minutes followed by blocking and permeabilization for 1 h in PBS with 1% BSA and 0.1% Triton X-100 at room temperature. Caco-2 cells were then incubated at 4°C with anti-NFkB p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in a humidified chamber and incubated with a FITC-conjugated goat anti-rabbit antibody at room temperature and with Hoechst 33342 (10 µg/mL) for 5 min.
For all immunofluorescence studies, slides were mounted with Vectashield Mounting Medium with DAPI (Vector laboratories, Ltd, UK). Fluorescence images from multiple fields were obtained using a Nikon Eclipse e 80i microscopy. The images were analyzed using NiS Elements D imaging software (Nikon Instruments Inc., NY, USA).