High-Resolution Three-Dimensional Imaging of Individual Astrocytes Using Confocal Microscopy.
High-Resolution Three-Dimensional Imaging of Individual Astrocytes Using Confocal Microscopy.
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DOI：
10.1002/cpns.92
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发表时间：
2020-03-01
影响因子：
--
通讯作者：
Reissner, Kathryn J
中科院分区：
文献类型：
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作者：
Testen, Anze;Kim, Ronald;Reissner, Kathryn J
星形胶质细胞在中枢神经系统中扮演着许多重要的角色。因此，确定星形胶质细胞在健康和疾病中的结构和功能特性是有益的。大多数检测星形胶质细胞形态的研究都使用了免疫分析方法来检测诸如胶质纤维酸性蛋白等标记物，这些标记物不足以概括这些细胞相当复杂的结构。在这里，我们描述了一种利用商业上可获得和验证的、遗传编码的星形胶质细胞膜相关荧光标记AAV5-GfaABC1D-LCK-GFP的方法。这种工具和方法允许对单个分离的星形胶质细胞的整体进行可视化，包括精细的外周突起。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，以获得详细的形态测量数据。我们进一步提供了一种免疫组织化学程序来评估分离的星形胶质细胞与突触标记在整个z平面的共定位。这项技术可以通过标准的实验室共聚焦显微镜和Imaris软件使用，允许详细分析固定组织中星形胶质细胞的形态和突触共定位。由John Wiley&Sons，Inc.实施。基本协议1：将AAV5-GfaABC1D-LCK-GFP显微注射到大鼠伏隔核内基本协议2：突触后密度的组织处理和免疫组织化学-95基本协议3：单细胞图像采集基本协议4：单细胞三维重建基本协议5：三维共定位分析。
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.