Antibodies for immunolabeling by light and electron microscopy: not for the faint hearted.
Antibodies for immunolabeling by light and electron microscopy: not for the faint hearted.
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DOI：
10.1007/s00418-014-1263-5
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发表时间：
2014-10
影响因子：
2.3
通讯作者：
Lucocq, John Milton
中科院分区：
文献类型：
--
作者：
Griffiths, Gareth;Lucocq, John Milton
关键词：
在细胞生物学家的武器库中，可靠的抗体是至关重要的工具，使用它们来定位免疫细胞化学抗原是它们最重要的应用之一。然而，抗体-抗原的相互作用比“锁与钥匙”的类比所暗示的要复杂和不可预测得多，而且试图证明抗体具有特异性的目标比人们通常认为的要困难得多。在这里，我们讨论了与一个非常复杂的问题相关的问题，即试图确定一种抗体（以及用它获得的结果）对光学或电子显微镜中使用的免疫标记方法是特异性的。我们讨论了越来越多的意识到，大量的商业抗体往往达不到所需的质量。我们提供了在免疫em中选择和测试抗体的指南。最后，我们描述了如何使用定量EM方法来识别抗体标记的可重复模式，并提取特定的标记分布。
Reliable antibodies represent crucial tools in the arsenal of the cell biologist and using them to localize antigens for immunocytochemistry is one of their most important applications. However, antibody–antigen interactions are much more complex and unpredictable than suggested by the old ‘lock and key’ analogy, and the goal of trying to prove that an antibody is specific is far more difficult than is generally appreciated. Here, we discuss the problems associated with the very complicated issue of trying to establish that an antibody (and the results obtained with it) is specific for the immunolabeling approaches used in light or electron microscopy. We discuss the increasing awareness that significant numbers of commercial antibodies are often not up to the quality required. We provide guidelines for choosing and testing antibodies in immuno-EM. Finally, we describe how quantitative EM methods can be used to identify reproducible patterns of antibody labeling and also extract specific labeling distributions.
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