Center for Neuroinflammation and Cardiometabolic Diseases, Georgia State University, Atlanta, GA 30302-5030, USA.
Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, FL 32611, USA.
STAR Protoc. 2022 Feb 10;3(1):101160. doi: 10.1016/j.xpro.2022.101160. eCollection 2022 Mar 18.
Here, we present a step-by-step protocol for three-dimensional reconstruction of astrocyte morphology, applied to the central amygdala oxytocin receptor-expressing astrocytes. This includes RNAse-free perfusion, combination of RNAscope and immunohistochemistry, and confocal imaging. This protocol provides detailed information about tissue handling and a comprehensive description of the RNAScope technique to label rat and mouse oxytocin receptor mRNA. We also describe three-dimensional reconstruction that allows the assessment of more than 70 different cellular parameters, powerful for studying astrocyte morphology and astrocyte-astrocyte interactions. For complete details on the use and execution of this protocol, please refer to Wahis et al. (2021) and Althammer et al. (2020).
在这里，我们提供了一个三步法的星形胶质细胞形态的三维重建方案，应用于表达催产素受体的大鼠杏仁中央核星形胶质细胞。该方案包括无 RNA 酶的灌流、RNAscope 和免疫组织化学的联合应用，以及共聚焦成像。该方案提供了关于组织处理的详细信息，并全面描述了用于标记大鼠和小鼠催产素受体 mRNA 的 RNAscope 技术。我们还描述了三维重建，它允许评估超过 70 种不同的细胞参数，对于研究星形胶质细胞形态和星形胶质细胞-星形胶质细胞相互作用非常有用。有关该方案使用和执行的完整详细信息，请参阅 Wahis 等人（2021 年）和 Althammer 等人（2020 年）的研究。
