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Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding
M. Sobol, VV. Philimonenko, P. Hozák
Language English Country Germany
Document type Comparative Study, Journal Article, Research Support, Non-U.S. Gov't
NLK
ProQuest Central
from 1997-01-01 to 1 year ago
Medline Complete (EBSCOhost)
from 2000-01-01 to 1 year ago
Nursing & Allied Health Database (ProQuest)
from 1997-01-01 to 1 year ago
Health & Medicine (ProQuest)
from 1997-01-01 to 1 year ago
Public Health Database (ProQuest)
from 1997-01-01 to 1 year ago
- MeSH
- Acrylic Resins diagnostic use MeSH
- Microscopy, Electron MeSH
- HeLa Cells ultrastructure MeSH
- Histological Techniques methods MeSH
- Immunohistochemistry methods MeSH
- Humans MeSH
- Freeze Substitution methods MeSH
- Preservation, Biological methods MeSH
- Pressure MeSH
- Tissue Embedding methods MeSH
- Freezing MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
In this study we present an optimized method of high-pressure freezing and automated freeze-substitution of cultured human cells, followed by LR White embedding, for subsequent immunolabeling. Also, the influence of various conditions of the freeze-substitution procedures such as temperature, duration, and additives in the substitution medium on the preservation of cryo-immobilized cells was analyzed. The recommended approach combines (1) automated freeze-substitution for high reproducibility and minimizing human-derived errors; (2) minimal addition of contrasting and fixing agents; (3) easy-to-use LR White resin for embedment; (4) good preservation of nuclei and nucleoli which are usually the most difficult structures to effectively vitrify and saturate in a resin; and (5) preservation of antigens for sensitive immunogold labeling.
References provided by Crossref.org
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- $a Sobol, Margarita $u Department of Biology of the Cell Nucleus, Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic, Prague, Czech Republic
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- $a Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding / $c M. Sobol, VV. Philimonenko, P. Hozák
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- $a In this study we present an optimized method of high-pressure freezing and automated freeze-substitution of cultured human cells, followed by LR White embedding, for subsequent immunolabeling. Also, the influence of various conditions of the freeze-substitution procedures such as temperature, duration, and additives in the substitution medium on the preservation of cryo-immobilized cells was analyzed. The recommended approach combines (1) automated freeze-substitution for high reproducibility and minimizing human-derived errors; (2) minimal addition of contrasting and fixing agents; (3) easy-to-use LR White resin for embedment; (4) good preservation of nuclei and nucleoli which are usually the most difficult structures to effectively vitrify and saturate in a resin; and (5) preservation of antigens for sensitive immunogold labeling.
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