High pressure freezing, electron microscopy, and immuno-electron microscopy of Tetrahymena thermophila basal bodies.

Janet B. Meehl, Thomas H. Giddings, Mark Winey

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)227-241
Number of pages15
JournalMethods in molecular biology (Clifton, N.J.)
Volume586
StatePublished - Dec 1 2009
Externally publishedYes

Fingerprint

Tetrahymena thermophila
Basal Bodies
Immunoelectron Microscopy
Freezing
Electron Microscopy
Freeze Substitution
Pressure
Electron Microscope Tomography
Cryopreservation
Green Fluorescent Proteins
Body Composition
Transmission Electron Microscopy
Plastics
Proteins
Staining and Labeling
Genes

ASJC Scopus subject areas

  • Medicine(all)

Cite this

High pressure freezing, electron microscopy, and immuno-electron microscopy of Tetrahymena thermophila basal bodies. / Meehl, Janet B.; Giddings, Thomas H.; Winey, Mark.

In: Methods in molecular biology (Clifton, N.J.), Vol. 586, 01.12.2009, p. 227-241.

Research output: Contribution to journalArticle

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