Almeida Vania W, Bates Margaret L, Bunge Mary Bartlett
The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, USA.
Department of Cell Biology, University of Miami Miller School of Medicine, Miami, FL, USA.
Methods Mol Biol. 2018;1739:195-212. doi: 10.1007/978-1-4939-7649-2_13.
The transmission electron microscope (TEM) enables a unique and valuable examination of cellular and extracellular elements in tissue in situ, in cultured cells, or in pellets derived from suspensions of cells or other materials such as nanoparticles. Here we focus on the preparation of cultured Schwann cells or Schwann cell-containing dorsal root ganglion cultures. To gain as life-like as possible views of the cellular details, it is imperative to achieve excellent preservation of the cellular structure. The steps in the preparation of cultures described in this chapter represent the results of many years of accumulated TEM images to find the best methods of preservation for Schwann cells, myelin, and basal lamina components. All the materials required are listed. The methods for fixing, dehydrating, and embedding a culture are described. Choosing an area in the culture to view, scoring it, cutting it out of the resin-embedded culture, mounting it appropriately for enface or cross-sectioning, and performing the semi-thin and thin sectioning are detailed. Explaining the way in which the sections are then stained for TEM completes the Methods section. Preservation of cultured Schwann cells and their myelin sheaths can be outstanding due to the direct and rapid but careful addition of the fixative solution to the culture dish.
透射电子显微镜（TEM）能够对组织原位、培养细胞或源自细胞悬浮液或其他材料（如纳米颗粒）的沉淀中的细胞和细胞外成分进行独特且有价值的检查。在这里，我们重点关注培养的雪旺细胞或含雪旺细胞的背根神经节培养物的制备。为了尽可能逼真地观察细胞细节，必须实现对细胞结构的出色保存。本章中描述的培养物制备步骤代表了多年积累的透射电子显微镜图像的结果，以找到保存雪旺细胞、髓鞘和基膜成分的最佳方法。列出了所需的所有材料。描述了固定、脱水和包埋培养物的方法。详细说明了如何在培养物中选择一个区域进行观察、评分、从树脂包埋的培养物中切出该区域、适当地安装以进行表面或横截面切片以及进行半薄和超薄切片。解释切片随后用于透射电子显微镜染色的方式完成了方法部分。由于将固定液直接、快速但小心地添加到培养皿中，培养的雪旺细胞及其髓鞘的保存效果可能非常出色。
