Skip to main content

Immunoelectron Microscopy of Cryofixed Freeze-Substituted Mammalian Tissue Culture Cells

  • Protocol
  • First Online:
Immunoelectron Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 657))

  • 3007 Accesses

Abstract

Mammalian tissue cultured cells are widely used in cell biology research. Immunoelectron microscopy is a powerful technique to define the subcellular localization of targeted antigens in the cultured cells. Cryofixation is now generally accepted as the best initial fixation step, to preserve the cellular fine structures and antigenicity. This chapter covers the practical procedures for immunoelectron microscopy of cryofixed freeze-substituted mammalian tissue cultured cells processed by high-pressure freezing.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Dahl, R. and Staehelin, L. A. (1989) High-pressure freezing for the preservation of biological structure: theory and practice. J. Electron Microsc. Tech. 13, 165–174.

    Article  PubMed  CAS  Google Scholar 

  2. McDonald, K. L., Morphew, M., Verkade, P., and Müller-Reichert, T. (2007) Recent advances in high-pressure freezing: equipment- and specimen-loading methods. Methods Mol. Biol. 369, 143–173.

    Article  PubMed  CAS  Google Scholar 

  3. Sawaguchi, A., McDonald, K. L., Karvar, S., and Forte, J. G. (2002) A new approach for high-pressure freezing of primary culture cells: the fine structure and stimulation-associated transformation of cultured rabbit gastric parietal cells. J. Microsc. 208, 158–166.

    Article  PubMed  CAS  Google Scholar 

  4. Sawaguchi, A., Yao, X., Forte, J. G., and McDonald K. L. (2003) Direct attachment of cell suspensions to high pressure freezing specimen planchettes. J. Microsc. 212, 13–20.

    Article  PubMed  CAS  Google Scholar 

  5. Sawaguchi, A., Ide, S., Goto, Y., Kawano, J., Oinuma, T., and Suganuma, T. (2001) A simple contrast enhancement by potassium permanganate oxidation for Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution. J. Microsc. 201, 77–83.

    Article  PubMed  CAS  Google Scholar 

  6. Kellenberger, E., Durrenberger, M., Villiger, W., Carlemalm, E., Wurtz, M. (1987) The efficiency of immunolabel on lowicryl sections compared to theoretical predictions. J. Histochem. Cytochem. 35, 959–969.

    Article  PubMed  CAS  Google Scholar 

  7. Kramarcy, N. R. and Sealock, R. (1991) Commercial preparations of colloidal gold-antibody frequently contain free active antibody.J. Histochem. Cytochem. 39, 37–39.

    Article  PubMed  CAS  Google Scholar 

  8. Slot, J. W. and Geuze, H. J. (1985) A new method of preparing gold probes for multiple-labeling cytochemistry. Eur. J. Cell Biol. 38, 87–93.

    PubMed  CAS  Google Scholar 

  9. Frens, G. (1973) Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature 241, 20–22.

    CAS  Google Scholar 

  10. De Mey, J., Moeremans, M., Geuens, R., Nuydens, R., and Brabander, M. D. (1981) High resolution light and electron microscopic localization of tubulin with the IGS (immuno gold staining) method. Cell Biol. Int. Rep. 5, 889–899.

    Article  PubMed  Google Scholar 

  11. Griffiths, G., ed. (1993) Particle markers for immunoelectron microscopy. Fine Structure Immunocytochemistry. Springer-Verlag, Berlin, 279–306.

    Google Scholar 

  12. Kellenberger, E. (1991) The potential of cryofixation and freeze substitution: observations and theoretical considerations. J. Microsc. 161, 183–203.

    Article  PubMed  CAS  Google Scholar 

  13. Bendayan, M. and Zollinger, M. (1983) Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique. J. Histochem. Cytochem. 31, 101–109.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Sawaguchi, A. (2010). Immunoelectron Microscopy of Cryofixed Freeze-Substituted Mammalian Tissue Culture Cells. In: Schwartzbach, S., Osafune, T. (eds) Immunoelectron Microscopy. Methods in Molecular Biology, vol 657. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-783-9_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-783-9_14

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-782-2

  • Online ISBN: 978-1-60761-783-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics