Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy, and Live Cell Imaging.
Microtubules (MTs) are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labeling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high-pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography.
Department of Botany, The University of British Columbia, Vancouver, BC, Canada.
Katherine
K
Celler
Department of Botany, The University of British Columbia, Vancouver, BC, Canada.
Miki
M
Fujita
Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada.
Eiko
E
Kawamura
Department of Biology, University of Saskatchewan, Saskatoon, SK, Canada.
Chris
C
Ambrose
Functional Plant Biology, Institute of Botany, University of Innsbruck, Sternwartestraße 15, 6020, Innsbruck, Austria.
Klaus
K
Herburger
Functional Plant Biology, Institute of Botany, University of Innsbruck, Sternwartestraße 15, 6020, Innsbruck, Austria. Andreas.Holzinger@uibk.ac.at.
Andreas
A
Holzinger
Department of Botany, The University of British Columbia, Vancouver, BC, Canada.
Geoffrey O
GO
Wasteneys
D017360:Arabidopsis / Q000166:cytology*; D002470:Cell Survival; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005615:Freezing; D005976:Glutaral; D008164:Luminescent Proteins / Q000378:metabolism; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure*; D009992:Osmium; D011312:Pressure; D013194:Staining and Labeling; D016707:Tissue Fixation
D008164:Luminescent Proteins; D009992:Osmium; D005976:Glutaral
ARK1; Correlative light and electron microscopy; EB1; Electron tomography; GFP; Immunofluorescence; Kinesin; Live cell imaging; MAP4; MBD; MOR1; Microtubules