SpringerProtocols: Abstract: High Pressure Freezing, Electron Microscopy, and Immuno-Electron Microscopy of Tetrahymena thermophila Basal Bodies

12. High Pressure Freezing, Electron Microscopy, and Immuno-Electron Microscopy of Tetrahymena thermophila Basal Bodies

By: Janet B. Meehl¹, Thomas H. Giddings Jr.¹, Mark Winey¹

Abstract

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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.

Affiliation(s): (1) Department of MCD Biology, University of Colorado, Boulder, CO, USA

Book Title: Cytoskeleton Methods and Protocols

Series: Methods in Molecular Biology | Volume: 586 | Pub. Date: Oct-02-2009 | Page Range: 227-241 | DOI: 10.1007/978-1-60761-376-3_12

Subject: Cell Biology

Key Words: Tetrahymena thermophila - High pressure freezing - Freeze substitution - Electron microscopy - Immuno-electron microscopy - Electron tomography

References

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