Immunofluorescence Analysis of Tight Junctions

Caco-2Bbe1 monolayers were grown in 12-well plates on glass coverslips, and immunofluorescence was performed using a previous protocol [21 (link)]. In brief, cell monolayers were fixed with 10% paraformaldehyde at 4 °C overnight, permeabilized using 0.1% Triton X-100, and non-specific binding was then blocked with 3% bovine serum albumin (Sigma, St. Louis, MO, USA). Cells were then washed with cold PBS and incubated with rabbit anti-ZO-1 antibody (Life Technologies, Invitrogen, Mississauga, ON, Canada) and goat Alexa-fluor 488-conjugated anti-rabbit IgG antibodies (Life Technologies). Cells were then mounted onto glass slides and visualized using a Leica DMI6000B fluorescence microscope and a DFC 360FX camera lens (Leica Microsystems, Concord, ON, Canada). Images were taken at random five times per slide at 20x of original magnification.