Journal Article

A method for efficient observation of intracellular membranes of monolayer culture cells by quick-freeze and freeze-fracture electron microscopy

Akikazu Fujita, Toyoshi Fujimoto, Nami Ozato-Sakurai and Hitomi Suzuki

in Microscopy

Published on behalf of The Japanese Society of Microscopy

Volume 61, issue 6, pages 441-446
Published in print December 2012 | ISSN: 2050-5698
Published online January 2012 | e-ISSN: 2050-5701 | DOI: https://dx.doi.org/10.1093/jmicro/dfs063
A method for efficient observation of intracellular membranes of monolayer culture cells by quick-freeze and freeze-fracture electron microscopy

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We previously developed a method of analyzing the two-dimensional distribution of membrane lipids by combining quick-freezing and freeze-fracture replica labeling (QF–FRL). In principle, this method can be applied to any membrane, but in practice it is not easy to observe cytoplasmic organelles efficiently without ice crystal damage. In this paper, we report a modification of our method that circumvents this problem. In the modified method, cells are cultured on a gold foil scratched with sandpaper and quick-frozen according to a high-pressure freezing method. This technique enables the efficient observation of intracellular structures such as the nucleus, endoplasmic reticulum, Golgi apparatus and mitochondria. Specific labeling of phosphoinositide 4,5-bisphosphate was confirmed in the obtained freeze-fracture replica. The present QF–FRL method is easy to use and should expedite the analysis of intracellular lipid distribution.

Keywords: electron microscopy; lipid; freeze-fracture; high-pressure freezing; metal contact freezing

Journal Article.  2125 words.  Illustrated.

Subjects: Biological Sciences

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