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Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy
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SYSNO ASEP 0387826 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy Author(s) Sobol, Margaryta (UMG-J) RID
Philimonenko, Vlada (UMG-J)
Philimonenko, Anatoly (UMG-J)
Hozák, Pavel (UMG-J) RID, ORCIDSource Title Histochemistry and Cell Biology. - : Springer - ISSN 0948-6143
Roč. 138, č. 1 (2012), s. 167-177Number of pages 11 s. Language eng - English Country DE - Germany Keywords immuno-gold labeling ; automated freeze-substitution ; LR White ; immunohistochemistry ; high-pressure freezing Subject RIV EB - Genetics ; Molecular Biology R&D Projects KAN200520704 GA AV ČR - Academy of Sciences of the Czech Republic (AV ČR) 2B06063 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) LC545 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) GAP305/11/2232 GA ČR - Czech Science Foundation (CSF) CEZ AV0Z50520514 - UMG-J (2005-2011) UT WOS 000305222300013 DOI 10.1007/s00418-012-0931-6 Annotation Using quantitative evaluation of immuno-gold labeling and antigen content, we evaluated various automated freeze-substitution protocols used in preparation of biological samples for immunoelectron microscopy. Protein extraction from cryoimmobilized cells was identified as a critical point during the freeze-substitution. The loss of antigens (potentially available for subsequent immuno-gold labeling) was not significantly affected by freezing, while the cryosubstitution with an organic solvent caused a significant loss of antigens. While addition of water can improve visibility of some cell structures, it strengthened the negative effect of cryosubstitution on antigen loss by extraction. This was, however, significantly reversed in the presence of 0.5% glutaraldehyde in the substitution medium. Furthermore, we showed that the level of these changes was antigen-dependent. In conclusion, low concentrations of glutaraldehyde can be generally recommended for cryosubstitution rather than the use of pure solvent, but the exact conditions need to be elaborated individually for certain antigens. Workplace Institute of Molecular Genetics Contact Nikol Škňouřilová, nikol.sknourilova@img.cas.cz, Tel.: 241 063 217 Year of Publishing 2013
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