Electron Microscopic Localization of in Situ Hybrids

  • Sandya Narayanswami
  • Barbara A. Hamkalo
Part of the Methods in Molecular Biology™ book series (MIMB, volume 29)


In situ hybridization is used to map the position of DNA or RNA sequences relative to cytologically-identifiable landmarks. Although major progress has been made in sequence mapping at the light microscope (LM) level, there are inherent limits to resolution, notably when sequences are closely linked or when the target structure is very small. Mapping in such situations is facilitated by exploiting the resolution of the electron microscope (EM). The principles underlying in situ hybridization are the same regardless of the nature of the label (i.e., radioactive or nonisotopic) and the level of analysis (i.e., LM or EM). Cytological preparations fixed to a solid support are denatured for DNA detection followed by incubation in an appropriate buffer containing a labeled nucleic acid probe and salt at an appropriate temperature to affect hybridization between the target and probe. Hybridization to RNA does not require the denaturation step. After hybridization and removal of unhybridized probe, hybrid sites are located by either autoradiography for radioactive probes or via tagging with a visible detector. This chapter focuses on the use of probes labeled in vitro with a variety of nonisotopic tags that can be detected based on high-affinity interactions with specific ligands, such as antibodies, and then visualized in the EM by colloidal gold particles. Except for the smallest colloids (1–5 nm in diameter), particles are quite electron dense, rendering them readily identifiable even at low magnification (i.e., 2000×)


Metaphase Chromosome Nick Translation Colloidal Gold Particle Fish Gelatin Centromeric Satellite 
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  1. 1.
    Rattner, J. B. and Hamkalo, B. A. (1978) Higher order structure in metaphase chromosomes. 1. The 250A fiber Chromosoma (Berl) 69, 363–372CrossRefGoogle Scholar
  2. 2.
    Maniatis, T, Fritsch, E F, and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. pp 466,467Google Scholar
  3. 3.
    Landegent, J. E., Jansen in de Wal, N, Baan, R A., Hoeijmakers, J. H J, and van der Ploeg, M. (1984) The use of 2-acetylaminofluorene modified probes for the indirect hybndocy tochemical detection of specific nucleic acid sequences in microscopic preparations. Exp. Cell. Res. 153, 61–72PubMedCrossRefGoogle Scholar
  4. 4.
    Fuchs, R and Daune, M (1972) Physical studies on deoxyribonucleic acid after covalent binding of a carcinogen Biochemistry 11, 2659–2666PubMedCrossRefGoogle Scholar
  5. 5.
    de Waele, M, de Mey, J, Moeremans, M, de Brabander, M., and Van Camp, B. (1983) Immunogold staining method for the light microscopic detection of leucocyte cell surface antigens with monoclonal antibodies. J Histochem. Cytochem. 31(3), 376–381.PubMedGoogle Scholar
  6. 6.
    Birrell, B. C, Hedberg, K. K., and Griffith, O. H. (1987) Pitfalls of immunogold labelling: Analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy J Histochem. Cytochem. 35(8), 843–853PubMedGoogle Scholar
  7. 7.
    Narayanswami, S. Lundgren, K, and Hamkalo, B A (1989) Deoxyribonucleicacid sequence mapping on metaphase chromosomes by immunoelectron microscopy. Scanning Microscopy Intl. Suppl. 3, 65–76.Google Scholar
  8. 8.
    Holgate, C S., Jackson, P., Cowen, P. N., and Bird, C C. (1983) Immunogold-silver staining: a new method of immunostaining with enhanced sensitivity. J. Histochem. Cytochem 31, 938–944PubMedGoogle Scholar
  9. 9.
    Narayanswami, S and Hamkalo, B A. (1987) Hybridization to chromatin and whole chromosome mounts, in Electron Microscopy in Molecular Biology. A Practical Approach (Sommerville, J. and Scheer, U, eds.) IRL, Oxford, UK, pp. 215–232Google Scholar
  10. 10.
    Moyzis, R K., Buckingham, J. ML, Cram, L. S., Dani, M., Deaven, L. L., Jones, M D, Meyne, J, Ratliff, R C, and Wu, J. R. (1988) A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc Natl Acad Sci. USA 85, 6622–6626PubMedCrossRefGoogle Scholar
  11. 11.
    Narayanswami, S. and Hamkalo, B. A. (1991) DNA sequence mapping using electron microscopy GATA 8(1), 14–23.Google Scholar

Copyright information

© Humana Press Inc, Totowa, NJ 1994

Authors and Affiliations

  • Sandya Narayanswami
    • 1
  • Barbara A. Hamkalo
    • 1
  1. 1.Department of Molecular Biology and BiochemistryUniversity of CaliforniaIrvine

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