Fritschy Jean-Marc
Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
Eur J Neurosci. 2008 Dec;28(12):2365-70. doi: 10.1111/j.1460-9568.2008.06552.x.
Immunohistochemistry is a sensitive and versatile method widely used to investigate the cyto- and chemoarchitecture of the brain. It is based on the high affinity and selectivity of antibodies for a single epitope. However, it is now recognized that the specificity of antibodies needs to be tested in control experiments to avoid false-positive results due to non-specific binding to tissue components or recognition of epitopes shared by several molecules. This 'Technical Spotlight' discusses other pitfalls, which are often overlooked, although they can strongly influence the outcome of immunohistochemical experiments. It also recapitulates the minimal set of information that should be provided in scientific publications to allow proper evaluation and replication of immunohistochemical experiments. In particular, tissue fixation and processing can have a strong impact on antigenicity by producing conformational changes to the epitopes, limiting their accessibility (epitope masking) or generating high non-specific background. These effects are illustrated for an immunoperoxidase staining experiment with three antibodies differing in susceptibility to fixation, using tissue from mice processed under identical conditions, except for slight variations in tissue fixation. In these examples, specific immunostaining can be abolished depending on fixation strength, or detected only after prolonged postfixation. As a consequence, antibody characterization in immunohistochemistry should include their susceptibility towards fixation and determination of the optimal conditions for their use.
免疫组织化学是一种灵敏且通用的方法，广泛用于研究大脑的细胞结构和化学结构。它基于抗体对单一表位的高亲和力和选择性。然而，现在人们认识到，抗体的特异性需要在对照实验中进行测试，以避免由于与组织成分的非特异性结合或对几种分子共有的表位的识别而导致假阳性结果。本“技术聚焦”讨论了其他一些常常被忽视的陷阱，尽管它们会强烈影响免疫组织化学实验的结果。它还概述了科学出版物中应提供的最少信息集，以便对免疫组织化学实验进行适当的评估和重复。特别是，组织固定和处理可通过使表位产生构象变化、限制其可及性（表位掩盖）或产生高非特异性背景，从而对抗原性产生强烈影响。使用在相同条件下处理的小鼠组织（除了组织固定略有差异），对三种对固定敏感性不同的抗体进行免疫过氧化物酶染色实验，说明了这些影响。在这些例子中，特异性免疫染色可根据固定强度而被消除，或者仅在延长的后固定后才能检测到。因此，免疫组织化学中的抗体表征应包括它们对固定的敏感性以及确定其使用的最佳条件。
