Tucker Richard P, Gong Qizhi
Department of Cell Biology and Human Anatomy, University of California, Davis, CA, USA.
Methods Mol Biol. 2014;1082:217-33. doi: 10.1007/978-1-62703-655-9_15.
One of the first steps in studies of gene function is the spatiotemporal analysis of patterns of gene expression. Indirect immunohistochemistry is a method that allows the detection of a protein of interest by incubating a histological section with an antibody or antiserum raised against the protein and then localizing this primary antibody with a tagged secondary antibody. To determine the cellular source of a protein of interest, or if a specific antibody is not available, specific transcripts can be localized using in situ hybridization. A histological section is incubated with a labeled RNA probe that is complementary to the target transcript; after hybridization with the target transcript, the labeled RNA probe can be identified with an antibody. Here we describe materials and methods used to perform basic indirect immunohistochemistry and in situ hybridization on frozen sections through the developing chicken brain, emphasizing controls and potential problems that may be encountered.
基因功能研究的首要步骤之一是对基因表达模式进行时空分析。间接免疫组织化学是一种通过将组织切片与针对目标蛋白产生的抗体或抗血清孵育，然后用标记的二抗定位该一抗来检测目标蛋白的方法。为了确定目标蛋白的细胞来源，或者在没有特定抗体的情况下，可以使用原位杂交来定位特定转录本。将组织切片与与目标转录本互补的标记RNA探针孵育；与目标转录本杂交后，可用抗体识别标记的RNA探针。在这里，我们描述了用于对发育中的鸡脑冰冻切片进行基本间接免疫组织化学和原位杂交的材料和方法，重点介绍了对照以及可能遇到的潜在问题。
