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Rapid Embedding Methods into Epoxy and LR White Resins for Morphological and Immunological Analysis of Cryofixed Biological Specimens

Published online by Cambridge University Press:  19 November 2013

Kent L. McDonald
Kent L. McDonald*
Affiliation:
Electron Microscope Laboratory, University of California, Berkeley, CA 94720, USA
*
*Corresponding author. E-mail: [email protected]
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Abstract

A variety of specimens including bacteria, ciliates, choanoflagellates (Salpingoeca rosetta), zebrafish (Danio rerio) embryos, nematode worms (Caenorhabditis elegans), and leaves of white clover (Trifolium repens) plants were high pressure frozen, freeze-substituted, infiltrated with either Epon, Epon-Araldite, or LR White resins, and polymerized. Total processing time from freezing to blocks ready to section was about 6 h. For epoxy embedding the specimens were freeze-substituted in 1% osmium tetroxide plus 0.1% uranyl acetate in acetone. For embedding in LR White the freeze-substitution medium was 0.2% uranyl acetate in acetone. Rapid infiltration was achieved by centrifugation through increasing concentrations of resin followed by polymerization at 100°C for 1.5–2 h. The preservation of ultrastructure was comparable to standard freeze substitution and resin embedding methods that take days to complete. On-section immunolabeling results for actin and tubulin molecules were positive with very low background labeling. The LR White methods offer a safer, quicker, and less-expensive alternative to Lowicryl embedding of specimens processed for on-section immunolabeling without traditional aldehyde fixatives.

Keywords

electron microscopybiological specimen preparationresin infiltrationresin polymerizationcorrelative light and electron microscopyLR Whitehigh pressure freezingfreeze substitutionon-section immunolabelingrapid embedding methods
Type
Techniques, Software, and Instrumentation Development
Information
Microscopy and Microanalysis , Volume 20 , Issue 1 , February 2014 , pp. 152 - 163
Copyright
Copyright © Microscopy Society of America 2014 

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