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Freeze substitution in 3 hours or less: FREEZE SUBSTIUTION IN 3 HOURS OR LESS
Abstract
Freeze substitution is a process for low temperature dehydration and fixation of rapidly frozen cells that usually takes days to complete. New methods for freeze substitution have been developed that require only basic laboratory tools: a platform shaker, liquid nitrogen, a metal block with holes for cryotubes and an insulated container such as an ice bucket. With this equipment, excellent freeze substitution results can be obtained in as little as 90 min for cells of small volume such as bacteria and tissue culture cells. For cells of greater volume or that have significant diffusion barriers such as cuticles or thick cell walls, one can extend the time to 3 h or more with dry ice. The 3-h method works well for all manner of specimens, including plants and Caenorhabditis elegans as well as smaller samples. Here, we present the basics of the techniques and some results from Nicotiana leaves, C. elegans adult worms, Escherichia coli and baby hamster kidney tissue culture cells.
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“another method to consider for monitoring autophagy in yeast (and potentially in plants) is to count autophagic bodies by TEM using at least two time points [149].The advantage of this approach is that it can provide accurate information on flux even when the autophagosomes are abnormally small [150,151].”
From Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition) 1 (2021) · cites this paper as [151]
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“There are morphometric procedures to measure or estimate the size range and the number of spherical objects by profiles in sections [162]; however, such methods have been used in autophagy research only a few times [43,151,164,165].”
From Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition) 1 (2021) · cites this paper as [151]
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Research works printed but not linked: 22
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Metadata
- Authors
- McDONALD, K. L., & WEBB, R. I.
- Venue
- Journal of Microscopy
- Publisher
- Wiley
- Published
- 2011-08-09
- Volume
- 243
- Issue
- 3
- Pages
- 227-233
- ISSN
- 0022-2720
- Cited by
- 211
- References
- 0
- Language
- en
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