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Statements

Subject Item
n2:RIV%2F68378050%3A_____%2F12%3A00387826%21RIV13-GA0-68378050
rdf:type
skos:Concept n12:Vysledek
dcterms: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.
dcterms: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
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
skos:notation
RIV/68378050:_____/12:00387826!RIV13-GA0-68378050
n12:predkladatel
n13:ico%3A68378050
n3:aktivita
n7:P n7:Z
n3:aktivity
P(2B06063), P(GAP305/11/2232), P(KAN200520704), P(LC545), Z(AV0Z50520514)
n3:cisloPeriodika
1
n3:dodaniDat
n16:2013
n3:domaciTvurceVysledku
n9:6055419 n9:9671706 n9:6029876 n9:2939711
n3:druhVysledku
n20:J
n3:duvernostUdaju
n17:S
n3:entitaPredkladatele
n4:predkladatel
n3:idSjednocenehoVysledku
163804
n3:idVysledku
RIV/68378050:_____/12:00387826
n3:jazykVysledku
n10:eng
n3:klicovaSlova
immuno-gold labeling; automated freeze-substitution; LR White; immunohistochemistry; high-pressure freezing
n3:klicoveSlovo
n6:high-pressure%20freezing n6:automated%20freeze-substitution n6:immuno-gold%20labeling n6:immunohistochemistry n6:LR%20White
n3:kodStatuVydavatele
DE - Spolková republika Německo
n3:kontrolniKodProRIV
[3A4B80AC3C7B]
n3:nazevZdroje
Histochemistry and Cell Biology
n3:obor
n18:EB
n3:pocetDomacichTvurcuVysledku
4
n3:pocetTvurcuVysledku
4
n3:projekt
n5:KAN200520704 n5:GAP305%2F11%2F2232 n5:2B06063 n5:LC545
n3:rokUplatneniVysledku
n16:2012
n3:svazekPeriodika
138
n3:tvurceVysledku
Sobol, Margaryta Hozák, Pavel Philimonenko, Anatoly Philimonenko, Vlada
n3:wos
000305222300013
n3:zamer
n19:AV0Z50520514
s:issn
0948-6143
s:numberOfPages
11
n15:doi
10.1007/s00418-012-0931-6