Peptide immunocytochemistry

Abstract

The potentials and limitations of immunocytochemical techniques are discussed with special reference to the light and electron microscopical localization of secretory peptides. Particular attention is given to methods for preserving peptide antigenicity and tissue structure as well as to control procedures for establishing immunocytochemical specificity. Additionally, comparisons between existing immunocytochemical methods with respect to their sensitivity, reliability and specificity are made. It is emphasized that peptide antigens differ with respect to physicochemical properties and that therefore different antigenic regions, even of the same peptide chain, are variously affected by different fixation and pretreatment conditions. The meaning of immunocytochemical specificity is extensively discussed. It is emphasized that adverse tissue pretreatment conditions make specific immunocytochemistry impossible. The need for concerted use of staining controls and specificity (absorption) controls is underlined. Additionally, antibodies must be regarded as region- or site-specific detection reagents, rather than as truly peptide-specific reagents. Thus, experiments employing either a single antiserum or sets of antisera with the same region-specificities are never sufficient to unequivocally identify tissue-bound antigens. This relates to the fact that antibodies only bind to a small portion (usually 4--8 amino acid residues) of a peptide antigen. Hence, the relative uniqueness of that particular small site will determine the peptide-specificity of its corresponding antibody. Since most secretory peptides share extensive sequence similarities and since, conceivably, many peptides have yet to be isolated and sequenced, such uniqueness can not be assumed. A way out of this dilemma is given by the concerted use of sets of antibodies monospecific for several antigenic regions of the peptide to be studied. Even so, chemical extraction and characterization work is often necessary in order to establish the identity of the demonstrated antigen. Such a combined biochemical and region-specific immunocytochemical approach is of great value for accurately identifying cellular and subcellular sites of secretory peptides and, in selected instances, also for the study of receptor-binding and peptide translocation, processing and intracellular transport. Defined immunocytochemistry requires appropriate tissue pretreatment conditions, adequate control procedures, and pure, monospecific antibodies. These requirements are considered in detail. In addition, the properties of conventional immunocytochemical techniques are discussed in relation to the newly developed labelled antigen detection techniques. Finally, techniques for the simultaneous localization of multiple antigens at both the light and electron microscopic level are described.