About: Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy

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About: Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy Goto Sponge NotDistinct Permalink

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rdf:type	skos:Concept http://linked.opendata.cz/ontology/domain/vavai/Vysledek
Description	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. 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. (en)
Title	Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy (en)
skos:prefLabel	Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy (en)
skos:notation	RIV/68378050:_____/12:00387826!RIV13-GA0-68378050
http://linked.open...avai/predkladatel	Ústav molekulární genetiky AV ČR, v. v. i.
http://linked.open...avai/riv/aktivita	P Z
http://linked.open...avai/riv/aktivity	P(2B06063), P(GAP305/11/2232), P(KAN200520704), P(LC545), Z(AV0Z50520514)
http://linked.open...iv/cisloPeriodika	1
http://linked.open...vai/riv/dodaniDat	2013
http://linked.open...aciTvurceVysledku	Sobol, Margaryta Philimonenko, Anatolij Hozák, Pavel Philimonenko, Vlada
http://linked.open.../riv/druhVysledku	J - Článek v odborném periodiku
http://linked.open...iv/duvernostUdaju	S - Úplné a pravdivé údaje nepodléhající ochraně podle zvláštních právních předpisů
http://linked.open...titaPredkladatele	Ústav molekulární genetiky AV ČR, v. v. i.
http://linked.open...dnocenehoVysledku	163804
http://linked.open...ai/riv/idVysledku	RIV/68378050:_____/12:00387826
http://linked.open...riv/jazykVysledku	eng - angličtina
http://linked.open.../riv/klicovaSlova	immuno-gold labeling; automated freeze-substitution; LR White; immunohistochemistry; high-pressure freezing (en)
http://linked.open.../riv/klicoveSlovo	high-pressure freezing immunohistochemistry LR White automated freeze-substitution immuno-gold labeling
http://linked.open...odStatuVydavatele	DE - Spolková republika Německo
http://linked.open...ontrolniKodProRIV	[3A4B80AC3C7B]
http://linked.open...i/riv/nazevZdroje	Histochemistry and Cell Biology
http://linked.open...in/vavai/riv/obor	EB
http://linked.open...ichTvurcuVysledku	4 (xsd:int)
http://linked.open...cetTvurcuVysledku	4 (xsd:int)
http://linked.open...vavai/riv/projekt	Structure and irregularities of the nucleoskeleton: identification and diagnostics. Functional Organisation of the Cell New nanoparticles for ultrastructural diagnostics Functions of myosin I and its binding partners in the cell nucleus
http://linked.open...UplatneniVysledku	2012
http://linked.open...v/svazekPeriodika	138
http://linked.open...iv/tvurceVysledku	Hozák, Pavel Sobol, Margaryta Philimonenko, Anatoly Philimonenko, Vlada
http://linked.open...ain/vavai/riv/wos	000305222300013
http://linked.open...n/vavai/riv/zamer	Molekulárně genetické a buněčné základy klíčových biologických procesů: genová exprese, onkogeneze, replikace virů, imunita a vývoj organismů
issn	0948-6143
number of pages	11 (xsd:int)
http://bibframe.org/vocab/doi	10.1007/s00418-012-0931-6
is http://linked.open...avai/riv/vysledek of	Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy

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