Binding of Anti-Trifluoroacetyl Antibodies to Isolated Hepatocytes Observed by Digital Fluorescence Microscopy

Abstract

These experiments were designed to observe specific binding of fluorescein-conjugated FAB′2 secondary antibodies to epitopes on the surface of isolated hepatocytes. The hepatocytes were attached as monolayers on microscope cover slips and an antigenic adduct known to be formed during metabolism of halothane, - trifluoroacetyl-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, was exchanged into their surface. Then the monolayers of hepatocytes were incubated with primary rabbit antibodies specific for the trifluoroacetyl group. Each coverslip was mounted in a perfusion chamber on a fluorescence microscope and a set of digital fluorescence images was made. Then fluorescein-conjugated goat-anti-rabbit FAB′2 secondary antibodies were flowed over the monolayer, the perfusion chamber was washed with buffer, and a second set of digital fluorescence images was made. The difference of these two sets of images demonstrated intense fluorescene superimposed on the outline of the cells. This intense fluorescence was not observed in control experiments in which the primary antibodies were omitted.