Immunocytochemistry by electron spectroscopic imaging using well defined boronated monovalent antibody fragments

Contributing to the rapidly developing field of immunoelectron microscopy a new kind of markers has been created. The element boron, incorporated as v ery stable carborane clusters into different kinds of peptides, served as a marker detectable by electron spectroscopic imaging (ESI) - an electron mi croscopic technique with high-resolution potential. Covalently linked immunoreagents conspicuous by the small size of both anti gen recognizing part and marker moiety are accessible by using peptide conc epts for label construction and their conjugation with Fab' fragments. Due to a specific labeling of the free thiol groups of the Fab' fragments, the antigen binding capacity was not affected by the attachment of the markers and the resulting immunoprobes exhibited an elongated shape with the antige n combining site and the label located at opposite ends. The labeling densi ties observed with these reagents were found to be significantly higher tha n those obtained by using conventional colloidal gold methods. Combined with digital image processing and analysis systems, boron-based ES I proved to be a powerful approach in ultrastructural immunocytochemistry e mploying pre- and post-embedding methods.