GFP-fluorescence and indirect immunocytochemistry
CV-1 or 293T cells seeded on glass coverslips, and MDCKII cells seeded on glass coverslips or on Transwell-Clear polyester membranes (0.4 μm, Costar) were fixed in 4% formaldehyde for 20 minutes at room temperature followed by three washes in PBS containing 0.1 mM CaCl2 and 0.1 mM MgCl2 (PBS++). For GFP-fluorescence, slides were mounted in vectashield mounting medium (Vector Laboratories, Inc.) and analyzed on a Zeiss Axiophot fluorescence microscope equipped with an RT slider SPOT camera (Diagnostic Instruments, Inc.) using SPOT RT Software v3.4, or by confocal microscopy (MRC-1024 Laser Scanning Confocal Imaging System; Bio-Rad). For indirect immunocytochemistry, after fixation, quenching was performed by incubating the cells for 10 minutes at room temperature in PBS++ containing 50 mM NH4Cl. Cells were then permeabilized with 0.4% Triton-X-100 for 5–10 minutes at room temperature. Unspecific binding was blocked with 0.5% Blocking Reagent (Roche) in PBS++ for 30 minutes at room temperature. Subsequently, the slides were incubated with primary antibodies for 1 hour at room temperature. After washing the cells three times in PBS++, bound primary antibodies were detected with fluorescently labelled secondary antibodies (Molecular Probes) for 30 minutes at room temperature. Following three washes in PBS++, slides were mounted and analyzed as described for GFP-fluorescence.