Larsson L I
Prog Histochem Cytochem. 1981;13(4):1-85.
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.
本文讨论了免疫细胞化学技术的潜力和局限性，特别提及了分泌肽在光学和电子显微镜下的定位。文中特别关注了保存肽抗原性和组织结构的方法以及确立免疫细胞化学特异性的对照程序。此外，还对现有免疫细胞化学方法在敏感性、可靠性和特异性方面进行了比较。强调肽抗原在物理化学性质上存在差异，因此即使是同一肽链的不同抗原区域，在不同的固定和预处理条件下也会受到不同程度的影响。文中广泛讨论了免疫细胞化学特异性的意义。强调不良的组织预处理条件会使特异性免疫细胞化学无法进行。强调了同时使用染色对照和特异性（吸收）对照的必要性。此外，必须将抗体视为区域或位点特异性检测试剂，而非真正的肽特异性试剂。因此，仅使用单一抗血清或具有相同区域特异性的抗血清组进行的实验，永远不足以明确鉴定组织结合抗原。这是因为抗体仅与肽抗原的一小部分（通常为4 - 8个氨基酸残基）结合。因此，该特定小位点的相对独特性将决定其相应抗体的肽特异性。由于大多数分泌肽具有广泛的序列相似性，并且可以想象，许多肽尚未被分离和测序，因此不能假定这种独特性。解决这一困境的方法是联合使用针对待研究肽的多个抗原区域具有单特异性的抗体组。即便如此，为了确定所显示抗原的身份，通常仍需要进行化学提取和表征工作。这种生化与区域特异性免疫细胞化学相结合的方法对于准确识别分泌肽的细胞和亚细胞位点，以及在特定情况下研究受体结合、肽转运、加工和细胞内运输具有重要价值。明确的免疫细胞化学需要适当的组织预处理条件、充分的对照程序以及纯的、单特异性抗体。文中对这些要求进行了详细讨论。此外，还讨论了传统免疫细胞化学技术与新开发的标记抗原检测技术相关的特性。最后，描述了在光学和电子显微镜水平同时定位多种抗原的技术。
