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Free keywords:
Electron microscopy; Immunolabeling; Oligodendrocytes; Cytoskeleton; Cryopreparation; Central nervous system; Myelin; Chemical fication
Abstract:
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.
