Evilsizor Megan N, Ray-Jones Helen F, Lifshitz Jonathan, Ziebell Jenna
Department of Child Health, University of Arizona College of Medicine - Phoenix; BARROW Neurological Institute, Phoenix Children's Hospital.
Department of Child Health, University of Arizona College of Medicine - Phoenix; Department of Biology and Biochemistry, University of Bath.
J Vis Exp. 2015 May 12(99):e52293. doi: 10.3791/52293.
Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3'-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.
免疫组织化学是一种广泛应用于原位检测抗原的存在、位置和相对丰度的技术。本入门级方案描述了使用小胶质细胞和神经元成分标记物对啮齿动物脑组织进行免疫组织化学染色所需的试剂、设备和技术。具体而言，本文是分别通过针对Iba1和泛神经元的免疫组织化学对小胶质细胞和神经元进行荧光可视化的分步方案。荧光双标记对于在同一样本中定位多种蛋白质特别有用，它提供了一个机会，可以准确观察细胞类型、受体、配体和/或细胞外基质之间相互关系的相互作用，以及单个细胞内的蛋白质共定位。与其他可视化技术不同，除非采取适当的预防措施，否则免疫组织化学荧光染色强度在染色后的数周甚至数月内可能会降低。尽管有此限制，但在许多应用中，荧光双标记比诸如3,3'-二氨基联苯胺四盐酸盐（DAB）或碱性磷酸酶（AP）等替代方法更受青睐，因为荧光更节省时间，并且能够更精确地区分两种或更多种标记物。讨论内容包括故障排除提示和建议，以促进实验成功。
