Summary
Preservation of Tetrahymena thermophila basal body ultrastructure for visualization by transmission electron microscopy is improved by a combination of high pressure freezing (HPF) and freeze substitution (FS). These methods also reliably retain the antigenicity of cellular proteins for immuno-electron microscopy, which enables the precise localization of green fluorescent protein (GFP)-tagged and native basal body proteins. The plastic-embedded samples generated by these methods take full advantage of higher resolution visualization techniques such as electron tomography. We describe protocols for cryofixation, FS, immunolabeling, and staining. Suggestions for trouble shooting and evaluation of specimen quality are discussed. In combination with identification and manipulation of a rapidly expanding list of basal body-associated gene products, these methods are being used to increase our understanding of basal body composition, assembly, and function.
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Acknowledgments
Our work on Tetrahymena is supported by the NIH (RO1 GM074746) and the March of Dimes Birth Defects Foundation (#1-FY07–520).
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Meehl, J.B., Giddings, T.H., Winey, M. (2009). High Pressure Freezing, Electron Microscopy, and Immuno-Electron Microscopy of Tetrahymena thermophila Basal Bodies. In: Gavin, R. (eds) Cytoskeleton Methods and Protocols. Methods in Molecular Biology, vol 586. Humana Press. https://doi.org/10.1007/978-1-60761-376-3_12
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DOI: https://doi.org/10.1007/978-1-60761-376-3_12
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