Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding

0355818 - ÚMG 2011 RIV DE eng J - Journal Article
Sobol, Margaryta - Philimonenko, Vlada - Hozák, Pavel
Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding.
Histochemistry and Cell Biology. Roč. 134, č. 6 (2010), s. 631-641. ISSN 0948-6143. E-ISSN 1432-119X
R&D Projects: GA AV ČR KAN200520704; GA MŠMT 2B06063; GA MŠMT LC545
Institutional research plan: CEZ:AV0Z50520514
Keywords : cryofixation * automated freeze-substitution * LR White
Subject RIV: EB - Genetics ; Molecular Biology
Impact factor: 4.727, year: 2010

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.
Permanent Link: http://hdl.handle.net/11104/0194499