Combining Whole-Mount In Situ Hybridization with Neuronal Tracing and Immunohistochemistry

  • Jeremy S. Duncan
  • Karen L. Elliott
  • Jennifer Kersigo
  • Brian Gray
  • Bernd Fritzsch
Part of the Neuromethods book series (NM, volume 99)


Despite the presence of several markers to study the expression of genes (eGFP, LacZ), in situ hybridization remains one of the most powerful techniques to analyze gene expression. While this allows cellular identification of the expression of a single gene and, using fluorescent in situ hybridization two or occasionally more genes, it is often necessary to combine this technology with assays of neuronal projection/morphology, protein expression using antibody staining, and histology for cytological details. Since each task has certain levels of false negatives, combining them in a single preparation can compromise further correlative studies due to loss of fluorescence, loss of antigenic epitope, or loss of tissue morphology. We have designed a protocol that, when performed in sequence, will enable the researcher to combine several of these technologies in the same sample saving time and sparing expense. By combining neuronal tracing, whole-mount in situ hybridization, immunohistochemistry, and histology one can extract a maximal amount of data with limited loss in fidelity of each technique and optimal data superposition for a more complete understanding of phenotypes.

Key words

In situ hybridization Lipophilic dye Immunohistochemistry Neuronal tracing 



Confocal images were obtained at the University of Iowa Carver Center for Imaging. We thank the Office of the Vice President for Research (OVPR), University of Iowa College of Liberal Arts and Sciences (CLAS), and the P30 core grant for support (DC 010362). This work was in part supported by a NASA base grant (Bernd Fritzsch) and 1R43GM108470-01 (Gray, Fritzsch).


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Jeremy S. Duncan
    • 1
  • Karen L. Elliott
    • 2
  • Jennifer Kersigo
    • 2
  • Brian Gray
    • 3
  • Bernd Fritzsch
    • 2
  1. 1.Division of Otolaryngology and Department of Neurobiology and AnatomyUniversity of Utah School of MedicineSalt Lake CityUSA
  2. 2.The Department of Biology, College of Liberal Arts and SciencesUniversity of IowaIowa CityUSA
  3. 3.Molecular Targeting Technologies Inc.West ChesterUSA

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