Goat alexa fluor 488 conjugated anti rabbit igg antibodies

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.

Polarized cell monolayers were fixed in paraformaldehyde, permeabilized in 0.1% Triton X-100, blocked with 3% bovine serum albumin (Sigma, St. Louis, MO) and incubated with rabbit anti-ZO-1 antibody (Life Technologies) and goat Alexa fluor 488-conjugated anti-rabbit IgG antibodies (Life Technologies) as described previously8 (link). Cells were then mounted onto glass slides using ProLong mounting solution (Life Technologies) and imaged with a Leica DMI6000B fluorescence microscope and companion DFC 360FX camera (Leica Microsystems, Concord, Canada) with 20x magnification.