Preservation, Sectioning, and Staining of Schwann Cell Cultures for Transmission Electron Microscopy Analysis
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.
Key wordsSchwann cells Primary cultures Embedding Ultramicrotome sectioning Transmission electron microscopy
We are grateful for the cartoons prepared by Megan Marlow at the Miami Project to Cure Paralysis. The protocols, developed over many years at the Miami Project, depended primarily upon funding from NINDS (# 09923), the Miami Project to Cure Paralysis, and the Buoniconti Fund. MBB is the Christine E. Lynn Distinguished Professor of Neuroscience. The authors declare no conflicts of interest with the contents of this chapter.