近端结扎法检测死后人脑G蛋白偶联受体复合物

Q2 Neuroscience

Current Protocols in Neuroscience Pub Date : 2020-01-13 DOI:10.1002/cpns.86

Ying Zhu, Andrew J. Dwork, Pierre Trifilieff, Jonathan A. Javitch

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引用次数: 5

摘要

结合免疫学和分子生物学方法，基于抗体的接近结扎试验(PLA)已经使用了十多年来检测和量化蛋白质-蛋白质相互作用，蛋白质修饰和蛋白质原位表达，包括在脑组织中。然而，由于标本的性质及其特殊处理，将这项技术转移到人脑样本需要采取一些预防措施。本研究采用聚乳酸明场检测技术对人死后大脑中多巴胺D2受体和腺苷A2A受体的表达及其邻近性进行了评估，并建立了一种系统的随机抽样方法来定量聚乳酸信号。©2019 by John Wiley &基本方案1:PLA_BF的样品制备和切片基本方案2:脑组织PLA_BF染色基本方案3:图像采集和结果分析支持方案:Luxol快速蓝/甲酚紫染色

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Detection of G Protein-Coupled Receptor Complexes in Postmortem Human Brain by Proximity Ligation Assay

Combining immunological and molecular biological methods, the antibody-based proximity ligation assay (PLA) has been used for more than a decade to detect and quantify protein-protein interactions, protein modification, and protein expression in situ, including in brain tissue. However, the transfer of this technology to human brain samples requires a number of precautions due to the nature of the specimens and their specific processing. Here, we used the PLA brightfield detection technique to assess the expression of dopamine D2 receptor and adenosine A2A receptor and their proximity in human postmortem brains, and we developed a systematic random sampling method to help quantify the PLA signals. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Sample preparation and sectioning for PLA_BF

Basic Protocol 2: PLA_BF staining of brain tissue

Basic Protocol 3: Image acquisition and result analysis

Support Protocol: Luxol fast blue/cresyl violet staining

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Current Protocols in Neuroscience Neuroscience-Neuroscience (all)

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期刊介绍： Current Protocols in Neuroscience is a one-stop resource for finding and adapting the best models and methods for all types of neuroscience experiments. Updated every three months in all formats, CPNS is constantly evolving to keep pace with the very latest discoveries and developments. A year of these quarterly updates is included in the initial CPNS purchase price.