Immunofluorescence assay

The immunofluorescence assay for NF-κB nuclear translocation was performed according to a previously described method.²⁰ Cells or sections of colon were incubated with a rabbit monoclonal anti-p65 antibody (Santa Cruz Biotechnology, Paso Robles, CA, USA) overnight at 4 °C. The samples were then immunostained with an Alexa Fluor 488-conjugated anti-rabbit IgG (Life Technology, Carlsbad, CA, USA) for 1 h at 37 °C. The samples were then washed and mounted in medium containing 4',6-diamidino-2-phenylindole (DAPI) to counterstain the nuclei, after which they were imaged using a confocal laser-scanning microscope (UltraView, Perkin Elmer, Cambridge, UK).