Testen Anze, Kim Ronald, Reissner Kathryn J
Neuroscience Curriculum, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Department of Psychology and Neuroscience, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Curr Protoc Neurosci. 2020 Mar;91(1):e92. doi: 10.1002/cpns.92.
Astrocytes play numerous vital roles in the central nervous system. Accordingly, it is of merit to identify structural and functional properties of astrocytes in both health and disease. The majority of studies examining the morphology of astrocytes have employed immunoassays for markers such as glial fibrillary acidic protein, which are insufficient to encapsulate the considerable structural complexity of these cells. Herein, we describe a method utilizing a commercially available and validated, genetically encoded membrane-associated fluorescent marker of astrocytes, AAV5-GfaABC1D-Lck-GFP. This tool and approach allow for visualization of a single isolated astrocyte in its entirety, including fine peripheral processes. Astrocytes are imaged using confocal microscopy and reconstructed in three dimensions to obtain detailed morphometric data. We further provide an immunohistochemistry procedure to assess colocalization of isolated astrocytes with synaptic markers throughout the z-plane. This technique, which can be utilized via a standard laboratory confocal microscope and Imaris software, allows for detailed analysis of the morphology and synaptic colocalization of astrocytes in fixed tissue. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Microinjection of AAV5-GfaABC1D-Lck-GFP into the nucleus accumbens of rats Basic Protocol 2: Tissue processing and immunohistochemistry for post-synaptic density-95 Basic Protocol 3: Single-cell image acquisition Basic Protocol 4: Three-dimensional reconstruction of single cells Basic Protocol 5: Three-dimensional colocalization analysis.
星形胶质细胞在中枢神经系统中发挥着众多重要作用。因此，确定健康和疾病状态下星形胶质细胞的结构和功能特性是有价值的。大多数研究星形胶质细胞形态的实验都采用了针对胶质纤维酸性蛋白等标志物的免疫测定法，但这些方法不足以概括这些细胞相当复杂的结构。在此，我们描述了一种利用市售且经过验证的、基因编码的星形胶质细胞膜相关荧光标记物AAV5-GfaABC1D-Lck-GFP的方法。这个工具和方法能够完整地可视化单个分离的星形胶质细胞，包括其精细的外周突起。使用共聚焦显微镜对星形胶质细胞进行成像，并进行三维重建以获得详细的形态计量学数据。我们还提供了一种免疫组织化学方法，用于评估分离的星形胶质细胞与整个z平面上突触标志物的共定位。这种技术可通过标准实验室共聚焦显微镜和Imaris软件使用，能够对固定组织中星形胶质细胞的形态和突触共定位进行详细分析。© 2020 John Wiley & Sons, Inc. 基本方案1：将AAV5-GfaABC1D-Lck-GFP微注射到大鼠伏隔核中 基本方案2：突触后致密蛋白95的组织处理和免疫组织化学 基本方案3：单细胞图像采集 基本方案4：单细胞的三维重建 基本方案5：三维共定位分析。
