Enhanced resolution of membranes in cultured cells by cryoimmobilization and freeze-substitution

Microscopy Research and Technique
P WildB M Humbel

Abstract

Investigations of cellular processes demand immediate arresting of the process at any given time and excellent retention of cellular material and excellent visibility of membranes. To achieve this goal we used cryofixation to arrest cellular processes instantly and tested diverse freeze-substitution protocols. Madin-Darby kidney cells and Vero cells were grown on carbon-coated sapphire disks. For cryofixation the sapphire disks covered with a cell monolayer were injected with the aid of a guillotine into liquid propane or ethane or a mixture of both cooled by liquid nitrogen. Freezing of the cryogen was prevented by using a partially insulated cylinder and by vigorous stirring that results in a substantial decrement of the freezing point of the cryogen. Cell monolayers can be cryofixed successfully using the guillotine in a safety hood at ambient temperature and humidity or at 37 degrees C and 45% humidity. The freezing unit can also be placed in a laminar flow for working under biohazard conditions. For visualizing cell membranes at high contrast and high resolution, cells were substituted in the presence of various concentrations of glutaraldehyde and osmium tetroxide and the temperature was raised to diverse final temperatur...Continue Reading

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Citations

Mar 1, 2012·Histochemistry and Cell Biology·Stefan Hübner, Athina Efthymiadis
Apr 28, 2004·Molecular and Biochemical Parasitology·Alexander MathisErik C Boettger
Jan 30, 2002·Micron : the International Research and Review Journal for Microscopy·Peter WildMonika Engels
Apr 25, 2009·The Journal of Biological Chemistry·Iveta BottovaSabrina Sonda
Mar 14, 2008·Journal of Virology·Anna Paula de OliveiraCornel Fraefel
Dec 23, 2004·Journal of Virology·Peter WildPaul Walther
Sep 29, 2005·Journal of Virology·Helene LeuzingerPeter Wild
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Mar 4, 2021·Journal of Phycology·Dovilė BarcytėMarek Eliáš

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