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In Situ Molecular Hybridization Techniques for Ultrathin Sections

  • Jean-Guy Fournier
  • Françoise Escaig-Haye
Part of the Methods in Molecular Biology™ book series (MIMB, volume 117)

Abstract

Several methods in molecular biology have now found a wide application in the morphological science domain allowing in situ detection of nucleic acids. It first became possible to visualize molecules in their natural environment 30 years ago, by adaptating nucleic acid hybridization techniques to histocyto-logical preparations (1). To date, many other molecular procedures whether or not derived from hybridization techniques can be used for in situ applications: polymerase chain reaction (2), nick-translation (3), tranferase-end labeling (4), and reverse transcription (5). Thus, the in situ hybridization technique has opened a new field of research that we can refer to as molecular histology (6). The technique of combining the molecular approach at the histological level is a powerful tool and is now widely used to examine the anatomical analysis of gene function. However, though it is possible to identify which type of cell contains nucleic acid sequences of interest and in which specific region of the cell they are preferentially found (7-12), in situ hydridization does not permit observation of the exact position of nucleic acid molecules in relation to the fine structure of the cell and its organelles. Such analysis is only possible if one is able to observe molecule detection with a high resolution. In order to do this, in the last 10 years, several efforts have been made to adapt in situ hybridization to electron microscopy (EM). This task was greatly helped by the development of nonisotopic labeled probes such as those which have incorporated biotinylated nucleotides (13,14). These allow for rapid and high resolution visualization of detected molecules, especially when the biotin is identified directly with ligands or antibodies conjugated to gold particles. Whatever the ultrastructural strategies used: preembedding (15-17), postembedding (18-21), cryoultramicrotomy(22), or whole-mounted cell deposited on grids (23-25), they should all follow the fundamental rules of the molecular hybridization reaction. This consists of establishing-in appropriate conditions-abase specific association according to the Watson-Crick criteria, between the nucle-otide sequences of the genetic probe and the complementary sequences present within the cell. The aim here is to obtain a high sensitivity of that reaction combined with good cell ultrastructural preservation. Cryoultramicrotomy fulfills the first criterion, but with poor cell integrity, whereas the preembedding protocol provides a high quality of morphology, but is associated with a low sensitivity of hybridization reaction. The third postembedding approach offers a compromise in which detection sensitivity and morphology preservation are acceptable (26). In this procedure, the use of acrylate-methacrylate hydro-soluble resins is a prerequisite for the successful hybridization of the nucleic acid sequences exposed only at the surface of the ultrathin sections. Among the resins available, Lowicryl K4M is the most popular, but other Lowicryl types such as HM20 (27) and K1 1M (28) or acrylic resins such as LR Gold (29), LR White (30), and Unicryl (31) are also efficient in permitting in situ molecular hybridization at the electron microscope level. The choice of the resin should be oriented to the one providing the best morphology of the material used.

Keywords

Gold Particle Microcentrifuge Tube Dextran Sulfate Nucleic Acid Sequence Hybridization Reaction 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Gall, J. C. and Pardue, M. L. (1969) Formation and detection of RNA-DNAhybrid molecules to cytological preparations. Proc. Natl. Acad. Sci. USA 63, 378–383.PubMedCrossRefGoogle Scholar
  2. 2.
    Nuovo, G. J. (1992) PCR in situ hybridization, Raven, New York.Google Scholar
  3. 3.
    Wijsman, J. H., Jonker, R. R., Keijzer, R., Van de velde, C. J., Cornelisse, C. J., and Van Dierendonck, J. H. (1993) Anew method to detect apoptosis in paraffin sections: in situ end labelling of fragmented DNA. J. Histochem. Cytochem. 41, 7.PubMedGoogle Scholar
  4. 4.
    Gorczyca, W., Bruno, S., Darzynkiewicz, R., Gonj, J., and Darzynkiewicz, Z. (1992) DNA strand breaks occurring during apopotosis their early in situ detection by terminal deoxynucleotidyl transferase and nick-translation assays and prevention by serine protease inhibitors. Int. J. Oncol. 1, 639–648.Google Scholar
  5. 5.
    Longley, J., Merchant, M. A., and Kacinski, B. M. (1989) In situ transcription and detection of CD 1a mRNA in epidermal cells: an alternative to standard in situ hybridization techniques. J. Invest. Dermatol. 93,432.PubMedCrossRefGoogle Scholar
  6. 6.
    Fournier, J. G. (1994) Molecular Histology. Lavoisier TEC and DOC, Paris, France.Google Scholar
  7. 7.
    Fournier, J. G., Kessous, A., Tiollais, P., Simard, R., Brechot, C., and Bouteille, M. (1982) Hepatitis B virus genome expression detected by in situ hybridization in a human hepatoma cell line. Biol. Cell 44, 197–200.Google Scholar
  8. 8.
    Fournier, J. G., Rozenblatt, S., and Bouteille, M. (1983) Localization of measles virus acid nucleic sequences in infected cells by in situ hybridization. Biol. Cell. 49, 287–290.PubMedGoogle Scholar
  9. 9.
    Lawrence, J. B. and Singer, R. H. (1986) Intracellular localization of messenger RNAs for cytoskeletal proteins. Cell 45,407–415.PubMedCrossRefGoogle Scholar
  10. 10.
    Meyer, J. L., Fournier, J. G., and Bouteille, M. (1986) Expression of integrated hepatitis B virus DNA in PLC/PRF/5, Hep 3B, and L6EC3 cell lines detected by in situ hybridization. Med. Biol. 64, 367–371.PubMedGoogle Scholar
  11. 11.
    Lawrence, J. B., Singer, R. H., and Marselle, L. M. (1989) Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell 57,493–502.PubMedCrossRefGoogle Scholar
  12. 12.
    Fournier, J. G., Prevot, S., Audoin, J., Letourneau, A., and Diebold, J. (1990) Fine analysis of HIV-1 RNA detection by in situ hybridization, in Modern Pathology of AIDS and Other Retroviral Infections Haase, H., Gluckmann, J. C., and Racz, P., eds.), Karger, Basel, pp. 62–68.Google Scholar
  13. 13.
    Langer, P. R., Waldrop, A. A., and Ward, D. C. (1981) Enzymatic synthesis of biotin-labelled polynucleotides novel nucleic acid affinity probes, Proc. Natl. Acad. Sci. USA 78, 6633–6637.PubMedCrossRefGoogle Scholar
  14. 14.
    Singer, R. H. and Ward, D. C. (1982) Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with biotynilated nucleotide analogue. Proc. Natl. Acad. Sci. USA 79, 7331–7335.PubMedCrossRefGoogle Scholar
  15. 15.
    Trembleau, A., Calas, A., and Fevre-Montange, M. (1990) Ultrastructural localization of oxytocin mRNA in the rat hypothalamus by in situ hybridization using a synthetic oligonucleotide. Mol. Brain Res. 8, 37–45.PubMedCrossRefGoogle Scholar
  16. 16.
    Guitteny, A. F. and Bloch, B. (1989) Ultrastructural detection of the vasopres-sine messenger RNA in the normal and Brattleboro rat. Histochemistry 92, 277–281.PubMedCrossRefGoogle Scholar
  17. 17.
    Tong, Y., Zhao, H., Simard, J., Labri, F., and Pelletier, G. (1989) Electron microscopic autoradiographc localization of prolactin mRNA in a rat pituitary. J. Histochem. Cytochem. 37, 567–571.PubMedGoogle Scholar
  18. 18.
    Binder, M., Tourmente, S., Roth, J., Rebaud, M., and Gehring, W. J. (1986) In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M embedded tissue using biotinylated probes and protein A-gold complexes.J. CellBiol. 102,1646–1653.CrossRefGoogle Scholar
  19. 19.
    Escaig-Haye, F., Grigoriev, V., and Fournier, J. G. (1989) Detection ultrastructurale d’ARN ribosomal par hybridation in situ à l’aide d’une sonde biotinylée sur coupes ultrafines de cellules animales en culture. C. R. Acad. Sci., Paris, D 429, 429–434.Google Scholar
  20. 20.
    Thiry, M. and Thiry-Blaise, L. (1989) In situ hybridization at the electron microscope. Improved method for precise localization of ribosomal DNA and RNA. Eur. J. CellBiol. 50,235–243.Google Scholar
  21. 21.
    Puvion-Dutilleul, F. and Puvion, E. (1989) Ultrastructural localization of viral DNA in thin sections of herpes simplexe virus type 1 infected cells by in situ hybridization. Eur. J. Cell Biol. 49, 99–109.Google Scholar
  22. 22.
    Morel, G., DM, E, and Gossard, F. (1989) Ultrastructural distribution of GH mRNA and GH intron I sequences in rat pituitary gland: effects of GH releasing factor and somatostatin. Mol. Cell Endocrinol. 65, 81–90.PubMedCrossRefGoogle Scholar
  23. 23.
    Hutchinson, N., Langer-Safer, P., Ward, D., and Hamkalo, B. (1982) In situ hybridization at the electron microscope level:hybrid detection by autoradiography and colloidal gold. J. CellBiol. 95, 609–618.CrossRefGoogle Scholar
  24. 24.
    Radic, M. Z., Lundgren, G., and Hamkalo, B. A. (1987) Curvature of mouse satellite DNA and condensation of heterochromatine. Cell 50, 1101–1108.PubMedCrossRefGoogle Scholar
  25. 25.
    Singer, R. H., Langevin, G. L., and Lawrence, J. B. (1989) Ultrastructural visualization of cytoskeletal messenger RNAs and their associated protein using double label in situ hybridization. J. CellBiol. 108, 2343–2353.CrossRefGoogle Scholar
  26. 26.
    Leguellec, D., Trembleau, A., Pechoux, C., and Morel, G. (1992) Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin sections vs post-embedding. J. Histochem. Cytochem. 40, 979–986.Google Scholar
  27. 27.
    Fournier, J. G. and Escaig-Haye, F. (1993) Electron microscopy of rRNA in situ hybridization on Lowicryl sections, in Hybridization Techniques for Electron Microscopy (Morel, G., ed.), CRC, Boca Raton, FL, pp. 243–268.Google Scholar
  28. 28.
    Leguellec, D., Frappart, L., and Desprez, P. Y. (1991) Ultrastructural localization of mRNA encoding for the EGF receptor in human breast cell cancer line BT 20 by in situ hybridization. J. Histochem. Cytochem. 39, 1–6.Google Scholar
  29. 29.
    Mc Fadden, G. I., Cornish, E. C., Boning, I., and Clarke, A. E. (1988) A simple fixation and embedding method for use in situ hybridization histochemistry of plant tissues. Histochem. J. 20, 575–586.CrossRefGoogle Scholar
  30. 30.
    Wenderroth, M. A. and Eisenberg, B. R. (1991) Ultrastructural distribution of myo-sin heavy chain mRNA in cardiac tissue: a comparison of frozen and LR White embedment. J. Histochem. Cytochem. 39, 1025–1033.Google Scholar
  31. 31.
    Cenacchi, G., Musiani, M., Gentilomi, G., Righi, S., Zerbini, M., Chandler, J. G., Scala, C., La Placa, M., and Martinelli, G. N. (1993) In situ hybridization at the ultrastructural level: localization of cytomegalovirus DNA using digoxigenin labelled probes. J. Submicrosc. Cytol. Pathol. 25, 341–345.PubMedGoogle Scholar
  32. 32.
    Escaig-Haye, F., Grigoriev, V., Peranzi, G., Lestienne, P., and Fournier, J. G. (1991) Analysis of human mitochondrial transcripts using electron microscopic in situ hybridization. J. Cell Sci. 100, 851–862.PubMedGoogle Scholar
  33. 33.
    Tourmente, S., Savre-Train, I., Berthier, F., and Renaud, M. (1990) Expression of six mitochondrial genes during Drosophila oogenesis: analysis by in situ hybridization. CellDiff. Dev. 31,137–149.Google Scholar
  34. 34.
    Lecher, P., Petit, N., Beziat, F., and Alziari, S. (1996) Localization by ultrastructural in situ hybridization of mitochondrial transcript in epithelial cells of a Drosophila sub-obscuradeletion mutant. Eur. J. CellBiol. 71,423–427.Google Scholar
  35. 35.
    Puvion-Dutilleul, F. and Puvion, E. (1996) Non-isotopic electron microscope in situ hybridization for studying the functional sub-compartmentalization of the cell nucleus. Histochem. Cell Biol. 106, 59–78.PubMedCrossRefGoogle Scholar
  36. 36.
    Escaig-Haye, F., Grigoriev, V., Sharova, I. A., Rudneva, V., Buckrinskaya, A., and Fournier, J. G. (1992) Ultrastructural localization of HIV-1 RNAand core proteins: Detection using double immunogold labelling after in situ hybridization and immunocytochemistry. J. Submicrosc. Cytol. Pathol. 24,437–443.PubMedGoogle Scholar
  37. 37.
    Mandry, P., Murray, B. A., Rieke, L., Becker, H., and Hofler, H. (1993) Post-embedding ultrastructural in situ hybridization on ultrathin cryosections and LR white resin sections. Ultrastruct. Pathol. 17,185–194.PubMedCrossRefGoogle Scholar
  38. 38.
    Ukimura, A., Deguchi, H., Kitaura, Y., Fujioka, S., Hirasawa, M., Kawamura, K., and Hirai, K. (1997) Intracellular viral localization in murine coxsackievirus-B3 181 myocarditis. Ultrastructural study by electron microscopic in situ hybridization. Am. J. Pathol. 150, 2061–2074.PubMedGoogle Scholar
  39. 39.
    Carlemalm, E., Villiger, W., Hobot, J. A., Acetarin, J. D., and Kellenberger, E. (1985) Low temperature embedding with Lowicryl resins: two new formulations and some applications. J. Microsc. 140, 55–72.PubMedGoogle Scholar
  40. 40.
    Singer, R. H., Lawrence, J. B., and Rashtchian, R. N. (1987) Towards a rapid and sensitive in situ hybridization methodology using istopic and non-isotopic probe, in In Situ Hybridization: Application to the Central Nervous System (Valentino, K., Eberwin, J., and Barchas, J., eds.), Oxford University Press, New York, pp. 71–96.Google Scholar

Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  • Jean-Guy Fournier
    • 1
  • Françoise Escaig-Haye
  1. 1.Institut de MycologieHôpital Pitié-SalpêtrièreParisFrance

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