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Fluorescence in situ Hybridization (FISH) on Tissue Cryosections

  • Irina SoloveiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 659)

Abstract

Recent progress in the understanding of the spatial organization of nuclear functions owes a lot to fluorescence in situ hybridization (FISH) methodology. The majority of studies using this technology have been carried out using cultured cells. However, nuclear processes in whole organisms, may be to a notable degree, different from those in cultured cells and actually not similar across different tissues. Therefore, for better understanding of nuclear processes in ex vivo organismal material, it is necessary to study nuclear organization in sections of tissue. FISH on sections is still not common in nuclear biology studies mostly due to methodological problems. The protocol suggested in this chapter is based on several years experience in hybridizing different probes on cryosections of various tissues.

Key words

Interphase nucleus Tissue sections Cryosections Fluorescence in situ hybridization Directly labeled probe Immunostaining Antigen retrieval 

Notes

Acknowledgments

The author thanks Yana Feodorova and Süleyman Kösem who, as graduate students, carried out some of the immuno-FISH experiments described in this protocol and Mike Fessing (University of Bradford) for providing BAC DNA for Rps27. This work was supported by DFG grant SO1054/1.

References

  1. 1.
    Cremer, M., Grasser, F., Lanctot, C., Müller, S., Neußer, M., Zinner, R., Solovei, I., and Cremer, T. (2008) Multicolor 3D Fluorescence In Situ Hybridization for Imaging Interphase Chromosomes, in Methods in Molecular Biology: The Nucleus (Hancock, R., Ed.) pp 205–239, Humana Press.Google Scholar
  2. 2.
    Cremer, M., Müller, S., Köhler, D., Brero, A., and Solovei, I. (2007) in CSH Protocols pp doi: 10.1101/pdb.prot4723.
  3. 3.
    Solovei, I., and Cremer, M. (2010) Combination of Immunostaining and 3D-FISH on Cultured Cells, in Methods in Molecular Biology: FISH (Bridger J. M., V. E., Ed.) Humana Press.Google Scholar
  4. 4.
    Solovei, I., Walter, J., Cremer, M., Habermann, H., Schermelleh, L., and Cremer, T. (2002) FISH on Three-Dimensionally Preserved Nuclei, in FISH (Beatty B., Larson S. M., J. Squire, Ed.) pp 119–157, Oxford University Press, Oxford.Google Scholar
  5. 5.
    Solovei, I., Grasser, F., and Lanctôt, C. (2007) in CSH Protocols pp doi: 10.1101/pdb.prot4729.
  6. 6.
    Shi, S. R., Cote, R. J., and Taylor, C. R. (1997) Antigen retrieval immunohistochemistry: past, present, and future. J Histochem Cytochem 45, 327–43.PubMedCrossRefGoogle Scholar
  7. 7.
    Shi, S. R., Cote, R. J., and Taylor, C. R. (2001) Antigen retrieval techniques: current perspectives. J Histochem Cytochem 49, 931–7.PubMedCrossRefGoogle Scholar
  8. 8.
    Henegariu, O., Bray-Ward, P., and Ward, D. C. (2000) Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling. Nat Biotechnol 18, 345–8.PubMedCrossRefGoogle Scholar
  9. 9.
    Müller, S., Neusser, M., Köhler, D., and Cremer, M. (2007) in CSH Protocols pp doi: 10.1101/pdb.prot4730.
  10. 10.
    Nimmakayalu, M., Henegariu, O., Ward, D. C., and Bray-Ward, P. (2000) Simple method for preparation of fluor/hapten-labeled dUTP. Biotechniques 28, 518–22.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Institute of Human Genetics, Biozentrum (LMU)University of MünichPlanegg-MartinsriedGermany

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