Nonfluorescent RNA In Situ Hybridization Combined with Antibody Staining to Visualize Multiple Gene Expression Patterns in the Embryonic Brain of Drosophila

  • David Jussen
  • Rolf UrbachEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2047)


In Drosophila, the brain arises from about 100 neural stem cells (called neuroblasts) per hemisphere which originate from the neuroectoderm. Products of developmental control genes are expressed in spatially restricted domains in the neuroectoderm and provide positional cues that determine the formation and identity of neuroblasts. Here, we present a protocol for nonfluorescent double in situ hybridization combined with antibody staining which allows the simultaneous representation of gene expression patterns in Drosophila embryos in up to three different colors. Such visible multiple stainings are especially useful to analyze the expression and regulatory interactions of developmental control genes during early embryonic brain development. We also provide protocols for wholemount and flat preparations of Drosophila embryos, which allow a more detailed analysis of gene expression patterns in relation to the cellular context of the early brain (and facilitate the identification of individual brain neuroblasts) using conventional light microscopy.


Drosophila Embryonic brain Neuroectoderm Neuroblast identification In situ hybridization Antibody staining 



The authors thank Janina Seibert and Dagmar Volland for sharing protocols and considerable expertise, Janina Seibert for Fig. 2c, and Karoline F. Kraft for critically reading the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (UR163/2-1 and UR163/3-1).


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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Institute of GeneticsUniversity of MainzMainzGermany

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