Immunofluorescence Analysis of Tight Junction Proteins

Epithelial cell layers were stained using the following primary antibodies: ZO-1 (1:100; BD Biosciences, NJ, USA). The secondary antibodies used were Alexa Fluor 488 goat anti-mouse or rabbit IgG, and Alexa Fluor 594 goat anti-mouse or rabbit IgG (1:500; Thermo Fisher Scientific, MA, USA). To determine occludin expression and cellular distribution, an occludin mouse monoclonal antibody (OC-3F10) was used as an Alexa Fluor® 594 Conjugate (Thermofischer). Nuclei were stained using DAPI (4′,6-Diamidin-2-phenylindol, conc. 1:2000). Immunofluorescence staining was analyzed by confocal laser scanning microscopy (LSM 780, Carl Zeiss, Jena, Germany) as previously described [9 (link),11 (link)].