Volpicelli-Daley Laura A, Levey Allan
Emory University, Atlanta, Georgia, USA.
Curr Protoc Neurosci. 2004 Feb;Chapter 1:Unit 1.2. doi: 10.1002/0471142301.ns0102s25.
The immunohistological methods described in this unit can be used to determine the precise localization of neurochemicals, receptors, and proteins throughout the nervous system. Determining the localization of a protein within defined brain nuclei and neuronal cell populations can provide important clues regarding its potential function. Immunoperoxidase reactions and light microscopy are commonly used to visualize the distribution of a single primary antibody directed to an antigen of interest. Double-labeling immunofluorescence and confocal microscopy techniques detect the localization of one protein relative to another protein and allow analysis of colocalization at a cellular and subcellular level. The colocalization of two proteins can also be quantified, allowing analysis of the extent of overlap between two labeled markers and measurements of changes in the localization of one protein relative to another following drug treatment or in animals that have been genetically modified. The theoretical limit of resolution of confocal microscopy is 0.1 to 0.2 microm.
本单元中描述的免疫组织学方法可用于确定神经化学物质、受体和蛋白质在整个神经系统中的精确定位。确定蛋白质在特定脑核和神经元细胞群体中的定位可为其潜在功能提供重要线索。免疫过氧化物酶反应和光学显微镜通常用于观察针对感兴趣抗原的单一一抗的分布。双标记免疫荧光和共聚焦显微镜技术可检测一种蛋白质相对于另一种蛋白质的定位，并允许在细胞和亚细胞水平分析共定位情况。两种蛋白质的共定位也可以进行量化，从而分析两个标记物之间的重叠程度，并测量药物治疗后或经过基因改造的动物中一种蛋白质相对于另一种蛋白质定位的变化。共聚焦显微镜的理论分辨率极限为0.1至0.2微米。
