High-Resolution Three-Dimensional Imaging of Individual Astrocytes Using Confocal Microscopy.

Q2 Neuroscience
Anze Testen, Ronald Kim, Kathryn J Reissner
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引用次数: 18

Abstract

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.

利用共聚焦显微镜对单个星形胶质细胞进行高分辨率三维成像。
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价值的。大多数检查星形胶质细胞形态的研究都采用免疫分析法检测诸如胶质纤维酸性蛋白等标记物,这些标记物不足以包涵这些细胞相当复杂的结构。在这里,我们描述了一种利用市售的和经过验证的、遗传编码的星形胶质细胞膜相关荧光标记物AAV5-GfaABC1D-Lck-GFP的方法。该工具和方法允许对单个分离的星形胶质细胞进行完整的可视化,包括精细的周围过程。星形胶质细胞成像使用共聚焦显微镜和三维重建,以获得详细的形态计量数据。我们进一步提供了一种免疫组织化学方法来评估分离的星形胶质细胞在整个z平面上与突触标记物的共定位。这项技术可以通过标准的实验室共聚焦显微镜和Imaris软件来使用,可以详细分析固定组织中星形胶质细胞的形态和突触共定位。©2020 by John Wiley & Sons, Inc。基本方案1:向大鼠伏隔核内显微注射AAV5-GfaABC1D-Lck-GFP基本方案2:突触后密度-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:单细胞三维重建基本方案5:三维共定位分析。
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来源期刊
Current Protocols in Neuroscience
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.
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