Abstract
Despite improvements in live imaging, fixation followed by embedding and sectioning for light or electron microscopy remains an indispensible approach in biology. During processing, small or delicate samples can be lost, damaged or poorly oriented. Here we present a protocol for overcoming these issues when, along with chemical fixation, the sample is fixed mechanically. The protocol features two alternatives for mechanical fixation: the sample is encased either in a rectangular block of agarose or between Formvar films suspended on a wire loop. We also provide methods for key steps all the way through to sectioning. We illustrate the method on the root of Arabidopsis thaliana, an object that is ∼0.15 mm in diameter and difficult to process conventionally. With this protocol, well-oriented sections can be obtained with excellent ultrastructural preservation. The protocol takes about 1 week.
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Acknowledgements
We thank T. Svitkina for electron microscopy expertise, and E.P. Eleftheriou and R.S. Poethig for technical assistance and comments. S.W. is partially supported by National Science Foundation grant 0920327 awarded to K.L.G. The Formvar loop method was developed with support from the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the United States Department of Energy through grant DE-FG-03ER15421 (to T.I.B).
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S.W., T.I.B. and K.L.G. developed the protocol and designed the experiments. S.W. performed the experiments. S.W., T.I.B. and K.L.G. wrote the manuscript.
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Supplementary information
Supplementary Video 1
Mechanical fixation: agarose method. (MOV 7548 kb)
Supplementary Video 2
Mechanical fixation: Formvar wire loop method. Green coated copper wire was used in the movie to decrease reflection and increase contrast for viewing. We recommend the use of bare copper wire in the actual experiments. (MOV 9336 kb)
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Wu, S., Baskin, T. & Gallagher, K. Mechanical fixation techniques for processing and orienting delicate samples, such as the root of Arabidopsis thaliana, for light or electron microscopy. Nat Protoc 7, 1113–1124 (2012). https://doi.org/10.1038/nprot.2012.056
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DOI: https://doi.org/10.1038/nprot.2012.056
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