Weil Marie-Theres, Ruhwedel Torben, Meschkat Martin, Sadowski Boguslawa, Möbius Wiebke
Max Planck Institute of Experimental Medicine, Department of Neurogenetics, Electron Microscopy Core Unit, Göttingen, Germany.
Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany.
Methods Mol Biol. 2019;1936:343-375. doi: 10.1007/978-1-4939-9072-6_20.
In this chapter, we describe protocols to study different aspects of oligodendrocytes and myelin using electron microscopy. First, we describe in detail how to prepare central nervous system tissue routinely by perfusion fixation of the animal and conventional embedding in Epon resin. Then, we explain how, with some modifications, chemically fixed tissue can be used for immunoelectron microscopy on cryosections. Chemical fixation and Epon embedding can also be applied to purified myelin to assess the quality of the preparation. Furthermore, we describe how cryopreparation by high-pressure freezing can be used to study the fine structure of myelin in nerve, brain, and spinal cord tissue. The differences in the structural appearance of oligodendrocytes and myelin between cryopreserved and conventionally processed samples are compared using representative images. Since primary cultured oligodendrocytes are used to study structure and function in vitro, we provide protocols for chemical fixation and Epon embedding of these cultures. Finally, we explain how the cytoskeleton of cultured oligodendrocytes can be visualized by using transmission electron microscopy on platinum-carbon replicas. In this chapter, we provide a wide range of protocols that can be applied to shed light on the different biological aspects of myelin and oligodendrocytes.
在本章中，我们描述了使用电子显微镜研究少突胶质细胞和髓鞘不同方面的实验方案。首先，我们详细描述了如何通过对动物进行灌注固定并常规包埋于Epon树脂中来制备中枢神经系统组织。然后，我们解释了经过一些修改后，化学固定的组织如何用于冷冻切片的免疫电子显微镜检查。化学固定和Epon包埋也可应用于纯化的髓鞘，以评估制备的质量。此外，我们描述了如何通过高压冷冻进行冷冻制备，以研究神经、脑和脊髓组织中髓鞘的精细结构。使用代表性图像比较了冷冻保存样本和传统处理样本中少突胶质细胞和髓鞘结构外观的差异。由于原代培养的少突胶质细胞用于体外研究结构和功能，我们提供了这些培养物的化学固定和Epon包埋方案。最后，我们解释了如何通过对铂 - 碳复制品进行透射电子显微镜观察来可视化培养的少突胶质细胞的细胞骨架。在本章中，我们提供了广泛的实验方案，可用于阐明髓鞘和少突胶质细胞的不同生物学方面。
