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)


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.


Gold Particle Microcentrifuge Tube Dextran Sulfate Nucleic Acid Sequence Hybridization Reaction 
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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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