In Situ Hybridization Combined with Retrograde Fluorescent Tract Tracing

  • Bibie M. Chronwall
  • Michael E. Lewis
  • James S. Schwaber
  • Thomas L. O’Donohue


In situ hybridization histochemistry is one of the most recent additions to neurobiological methods. In this method, a labeled DNA or RNA sequence is hybridized to its specific complementary messenger RNA (mRNA) in histological sections and visualized by autoradiography or histochemistry. The method originated in the field of molecular genetics and was originally used for localization of specific DNAs in metaphase chromosomes (Gall and Par-due, 1969; Jones and Robertson, 1970; Jacob et al., 1971). Later, globin mRNA was detected in dispersed mammalian cells (Harrison et al., 1973). The early studies are good sources for information on techniques and their application to the analysis of invertebrate development (Capco and Jeffery, 1978; An-gerer and Angerer, 1981; McAllister et al., 1983; Cox et al., 1984). This chapter focuses on the mammalian CNS. Most commonly, probes are radioactively labeled, but biotinylated probes have also been tried (Singer and Ward, 1982; Varndell et al., 1984; Binder et al., 1986). An advantage of using radioactively labeled probes is that grain counting will give a relative quantification of mRNA levels in specific cells (Szabo et al., 1977; Brahic and Haase, 1978; Griffin et al., 1985; Uhl and Sasek, 1986; Wilcox et al., 1986a,b; Young et al., 1986b; Chronwall et al., 1987). Compared to immunohistochemistry, in situ hybridization offers the advantage of localizing the anatomic site for protein synthesis, not merely detecting the presence of the protein.


Synthetic Oligonucleotide Nonspecific Hybridization Hybridization Histochemistry SITU Hybridization Percent Sucrose 
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Copyright information

© Springer Science+Business Media New York 1989

Authors and Affiliations

  • Bibie M. Chronwall
    • 1
  • Michael E. Lewis
    • 2
  • James S. Schwaber
    • 3
  • Thomas L. O’Donohue
    • 4
  1. 1.School of Basic Life Sciences, Division of Structure and Systems BiologyUniversity of MissouriKansas CityUSA
  2. 2.Cephalon Inc.West ChesterUSA
  3. 3.Neurobiology GroupE. I. DuPont de Nemours and Co., Inc.WilmingtonUSA
  4. 4.J. D. Searle & Co.CNS ResearchSt. LouisUSA

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