High pressure freezing, electron microscopy, and immuno-electron microscopy of Tetrahymena thermophila basal bodies.
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.
Meehl
Janet B
JB
Department of MCD Biology, University of Colorado, Boulder, CO, USA.
Giddings
Thomas H
TH
Winey
Mark
M
D000818:Animals; D002502:Centrioles / Q000378:metabolism* / Q000648:ultrastructure; D015925:Cryopreservation / Q000379:methods; D055032:Electron Microscope Tomography / Q000379:methods; D017110:Freeze Substitution; D005615:Freezing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008854:Microscopy, Electron / Q000379:methods*; D016253:Microscopy, Immunoelectron / Q000379:methods*; D011312:Pressure; D016808:Tetrahymena thermophila / Q000378:metabolism* / Q000648:ultrastructure; D016610:Tissue Embedding / Q000379:methods
D049452:Green Fluorescent Proteins