Staining Serial Thick Sections For High-Voltage Electron Microscopy
Ultrastructural studies often require three-dimensional reconstructions from serial electron micrographs. The use of thick sections and high voltage electron microscopy can reduce the numbers of serial sections needed, but we have found that over 300 serial sections (0.5 μm thick) are still needed for our studies of taste bud ultrastructure. Serial reconstructions are almost always done with sections mounted onto formvar-coated slot grids, because they provide an unobscured view of the specimen. Staining and carbon coating several hundred formvar-coated slot grids often results in the loss of serial sections because the formvar films break due to handling during the staining and carbon-coating procedures. In addition, the staining process itself can be tedious and time-consuming when staining thick sections because staining times of hours are often required. Such lengthy staining times may result in staining artifacts due to exposure of the stain to the atmosphere, especially with lead stains.