Hoffman Gloria E, Murphy Kelley J, Sita Luciane V
Department of Biology, Morgan State University, Baltimore, Maryland.
Department of Anatomy, University of São Paulo, São Paulo, Brazil.
Curr Protoc Neurosci. 2016 Jul 1;76:2.12.1-2.12.37. doi: 10.1002/cpns.1.
When using immunocytochemistry, investigators may not know how to optimize staining or how to troubleshoot the method when staining fails. Lacking are guides for comparing techniques and applying information derived from one staining method to another. Newer methods amplify signal detection, but will not necessarily work at the same primary antibody concentrations used for less sensitive reactions. Recommendations of optimal titers are often not accurate and are not usually accompanied by information on the method used to test those antibodies or the specifics of the assay. When the staining does not work, the investigators do not know how to determine if the antiserum is bad, the tissue is bad, or the method is inappropriate for their staining. This unit describes detailed procedures for determining optimal staining and applying that information to three common immunofluorescence methods. Lastly, a formula is provided for converting among the different methods. © 2016 by John Wiley & Sons, Inc.
在使用免疫细胞化学技术时，研究人员可能不知道如何优化染色，也不知道在染色失败时如何对该方法进行故障排除。目前缺乏比较不同技术以及将一种染色方法所得信息应用于另一种方法的指南。更新的方法增强了信号检测能力，但不一定能在用于敏感性较低反应的相同一抗浓度下发挥作用。关于最佳滴度的建议往往不准确，而且通常不附带用于测试这些抗体的方法或检测细节的信息。当染色不成功时，研究人员不知道如何确定是抗血清质量差、组织质量差，还是该方法不适用于他们的染色。本单元描述了确定最佳染色并将该信息应用于三种常见免疫荧光方法的详细程序。最后，提供了一个用于在不同方法之间进行转换的公式。© 2016 约翰威立国际出版公司
