BINDING OF ANTI-TRIFLUOROACETYL ANTIBODIES TO ISOLATED HEPATOCYTES OBSERVED BY DIGITAL FLUORESCENCE MICROSCOPY

These experiments were designed to observe specific binding of fluores cein-conjugated FAB'2 secondary antibodies to epitopes on the surface of isolated hepatocytes. The hepatocytes were attached as monolayers o n microscope cover slips and an antigenic adduct known to be formed du ring metabolism of halothane, tyl-1,2-dioleoyl-sn-glycero-3-phosphoeth anolamine, was exchanged into their surface. Then the monolayers of he patocytes were incubated with primary rabbit antibodies specific for t he trifluoroacetyl group. Each coverslip was mounted in a perfusion ch amber on a fluorescence microscope and a set of digital fluorescence i mages was made. Then fluorescein-conjugated goat-anti-rabbit FAB'2 sec ondary antibodies were flowed over the monolayer, the perfusion chambe r was washed with buffer, and a second set of digital fluorescence ima ges was made. The difference of these two sets of images demonstrated intense fluorescene superimposed on the outline of the cells. This int ense fluorescence was not observed in control experiments in which the primary antibodies were omitted. (C) 1994 Academic Press, Inc.