Cells were seeded in permanox chamber slides (Nalge Nunc International, Rochester, NY) at a density of 1610 5 cells/ml and incubated in a 5% CO 2 humidified chamber for 48-72 hours. Cells were washed three times in ice-cold PBS and fixed for 10 min in ice-cold acetone. Normal liver sections and frozen tumor sections were prepared as previously described [37] . Cells were blocked for 15 min with 1% normal goat serum (Sigma-Aldrich) in PBS and incubated for 30 min at room temperature with a 1:200 dilution of primary MAb 9.2 specific for CEACAM1-4S. After three washes in PBS, cultures were fixed with 4% paraformaldehyde for 1 min, quenched in 0.1 M glycine in PBS, and incubated at 4uC for 30 min in Alexa 488 conjugated goat anti-mouse IgG (1:400 dilution). Cells were examined by fluorescence microscopy using a Nikon Eclipse E800 microscope (Nikon Instruments, Inc., Melville, NY).