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

Details

Serval ID
serval:BIB_211CF528950F
Type
Article: article from journal or magazin.
Collection
Publications
Title
Enhanced resolution of membranes in cultured cells by cryoimmobilization and freeze-substitution.
Journal
Microscopy Research and Technique
Author(s)
Wild P., Schraner E.M., Adler H., Humbel B.M.
ISSN
1059-910X (Print)
ISSN-L
1059-910X
Publication state
Published
Issued date
2001
Volume
53
Number
4
Pages
313-321
Language
english
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 temperatures. Substitution for 4 hours at -90 degrees C in anhydrous acetone containing 0.25% anhydrous glutaraldehyde and 0.5% osmium tetroxide followed by a temperature rise of 5 degrees C/hour to 0 degrees C and final incubation for 1 hour at 0 degrees C resulted in high contrast and excellent visibility of subcellular components at the level of the membrane bilayer. The high spatial and temporal resolution makes this methodology an excellent tool for studying cell membrane-bound processes, such as virus-cell interactions.
Keywords
Animals, Cell Line, Cell Membrane/ultrastructure, Cell Nucleus/ultrastructure, Cercopithecus aethiops, Cryoprotective Agents, Freeze Substitution/methods, Temperature, Tissue Fixation, Vero Cells
Pubmed
Web of science
Create date
18/10/2012 14:11
Last modification date
20/08/2019 12:57
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