A Protocol for Double Fluorescent In Situ Hybridization and Immunohistochemistry for the Study of Embryonic Brain Development in Tribolium castaneum

  • Marita Buescher
  • Georg Oberhofer
  • Natalia Carolina Garcia-Perez
  • Gregor BucherEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2047)


The red flour beetle, Tribolium castaneum, is an emerging model system well suited to the study of embryonic brain development and evolution (see Chapters  11 and  13). Brain genesis is driven by specific gene products whose expression underlies a tight spatiotemporal control. Therefore, the analysis of gene expression in time and space provides valuable insights into the molecular mechanisms that govern brain development. Since Tribolium-specific antibodies are scarce, fluorescent RNA in situ hybridization is the method of choice to determine the dynamics of individual gene expression. We have modified common RNA in situ protocols to facilitate the concomitant detection of two gene-specific expression patterns (double fluorescent RNA in situ). In addition, we describe a procedure which combines fluorescent single RNA in situ and immunostaining with gene-specific antibodies. Conventional in situ using RNA probes that are complementary to mature mRNAs often produce diffuse signals. We demonstrate that RNA in situ probes complementary to intronic gene sequences facilitate single cell resolution because the fluorescent signal is restricted to the nucleus. We believe our protocols can be adapted easily to suit the analysis of brain development in other insect species.


Tribolium castaneum Brain development Gene expression pattern Fluorescent RNA in situ hybridization (FISH) Intron-specific probes Tyramide signal amplification (TSA) 


  1. 1.
    Brown SJ, Shippy TD, Miller S et al (2009) The red flour beetle, Tribolium castaneum (Coleoptera): a model for studies of development and pest biology. Cold Spring Harb Protoc 2009(8):pdb.emo126. Scholar
  2. 2.
    Biffar L, Stollewerck A (2014) Conservation and evolutionary modifications of neuroblast expression patterns in insects. Dev Biol 388:103–106CrossRefGoogle Scholar
  3. 3.
    Koniszewski ND, Kollmann M, Bigham M et al (2016) The insect central complex as model for heterochronic brain development-background, concepts and tools. Dev Genes Evol 226(3):209–219. Scholar
  4. 4.
    Panov AA (1959) Structure of the insect brain at successive stages of postembryonic development. II. The central body. Entomol Rev 38Google Scholar
  5. 5.
    Shippy TD, Coleman CM, Tomoyasu Y, Brown SJ (2009) Concurrent in situ hybridization and antibody staining in red flour beetle (Tribolium) embryos. Cold Spring Harb Protoc 2009(8):pdb.emo126. Scholar
  6. 6.
    Dönitz J, Schmitt-Engel C, Grossmann D, Gerischer L, Tech M, Schoppmeier M, Klingler M, Bucher G (2015) iBeetle-Base: a database for RNAi phenotypes in the red flour beetle Tribolium castaneum. Nucleic Acids Res:gku1054Google Scholar
  7. 7.
    Doenitz J, Gerischer L, Hahnke S et al (2018) Expanded and updated data and a query pipeline for iBeetle-Base. Nucleic Acids Res 46(D1):D831–D835. Scholar
  8. 8.
    Bobrow MN, Harris TD, Shaughnessy KJ et al (1989) Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J Immunol Methods 125(1–2):279–285CrossRefGoogle Scholar
  9. 9.
    Bobrow MN, Shaughtnessy KJ, Litt GJ (1991) Catalyzed reporter deposition, a novel method of signal amplification. II. Application to membrane immunoassays. J Immunol Methods 137(1):103–112CrossRefGoogle Scholar
  10. 10.
    Hopman AH, Ramaekers FC, Speel EJ (1998) Rapid synthesis of biotin-, dioxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for in situ hybridization using card amplification. J Histochem Cytochem 48(8):771–777CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Marita Buescher
    • 1
  • Georg Oberhofer
    • 1
  • Natalia Carolina Garcia-Perez
    • 1
  • Gregor Bucher
    • 1
    Email author
  1. 1.Department of Evolutionary Developmental Genetics, Johann-Friedrich-Blumenbach InstituteGZMB, University of GöttingenGöttingenGermany

Personalised recommendations