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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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Abstract

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.

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Acknowledgments

We thank Dr. Michael Hess, Dr. Andreas Käch, Dr. Kent McDonald, Dr. Bruno Humbel, Dr. Mary Morphew, Dr. Yannick Schwab, Dr. Heinz Schwarz, Dr. Danièle Spehner, Dr. Daniel Studer, Dr. Cveta Tomova, Dr. Paul Verkade (in alphabetical order) for very useful discussions about high-pressure freezing and freeze-substitution. We thank Karel Janoušek, Ivana Nováková, Iva Jelínková, and Pavel Kříž for their excellent technical help. This work was supported by the Academy of Sciences of the Czech Republic (reg. no. KAN200520704), Grants 2B06063 and LC545 of the Ministry of Education, Youth and Sports of the Czech Republic, by the Grant Agency of the Czech Republic (Grant P305/11/2232) and by the Institutional Grant IMG No. AV0Z50520514.

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Sobol, M.A., Philimonenko, V.V., Philimonenko, A.A. et al. Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy. Histochem Cell Biol 138, 167–177 (2012). https://doi.org/10.1007/s00418-012-0931-6

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