Počet záznamů: 1
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 Druh ASEP J - Článek v odborném periodiku Zařazení RIV J - Článek v odborném periodiku Poddruh J Článek ve WOS Název Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy Tvůrce(i) Sobol, Margaryta (UMG-J) RID
Philimonenko, Vlada (UMG-J)
Philimonenko, Anatoly (UMG-J)
Hozák, Pavel (UMG-J) RID, ORCIDZdroj.dok. Histochemistry and Cell Biology. - : Springer - ISSN 0948-6143
Roč. 138, č. 1 (2012), s. 167-177Poč.str. 11 s. Jazyk dok. eng - angličtina Země vyd. DE - Německo Klíč. slova immuno-gold labeling ; automated freeze-substitution ; LR White ; immunohistochemistry ; high-pressure freezing Vědní obor RIV EB - Genetika a molekulární biologie CEP KAN200520704 GA AV ČR - Akademie věd 2B06063 GA MŠMT - Ministerstvo školství, mládeže a tělovýchovy LC545 GA MŠMT - Ministerstvo školství, mládeže a tělovýchovy GAP305/11/2232 GA ČR - Grantová agentura ČR CEZ AV0Z50520514 - UMG-J (2005-2011) UT WOS 000305222300013 DOI 10.1007/s00418-012-0931-6 Anotace 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. Pracoviště Ústav molekulární genetiky Kontakt Nikol Škňouřilová, nikol.sknourilova@img.cas.cz, Tel.: 241 063 217 Rok sběru 2013
Počet záznamů: 1