Immunostaining of the Embryonic and Larval Drosophila Brain

  • Frank HirthEmail author
  • Danielle C. Diaper
Part of the Methods in Molecular Biology book series (MIMB, volume 2047)


Immunostaining is used to visualize the spatiotemporal expression pattern of developmental control genes that regulate the genesis and specification of the embryonic and larval brain of Drosophila. It is also used to visualize the effects of targeted misexpression or inactivation of disease-related genes. Immunostaining uses specific antibodies to mark expressed proteins and allows their localization to be traced. This method reveals insights into gene regulation, cell type specification, neuron and glial differentiation, axonal and synaptic scaffolding and posttranslational protein modifications underlying the patterning and specification of the maturing brain. Depending on the targeted protein, it is possible to visualize a multitude of regions of the Drosophila brain, such as small groups of neurons or glia, defined subcomponents of the brain’s axon scaffold, or pre- and postsynaptic structures of neurons. Thus, antibody probes that recognize defined tissues, cells, or subcellular structures like axons or synaptic terminals can be used as markers to identify and analyze phenotypes in embryos and larvae. Several antibodies, combined with different labels can be used concurrently to examine protein colocalization. This protocol spans over 3–4 days.


Drosophila Embryo Larva Brain Immunostaining Fluorescence immunocytochemistry Dissection Antibody 



This work was supported by the UK Medical Research Council (G0701498; MR/L010666/1), the Biotechnology and Biological Sciences Research Council (BB/N001230/1), the MND Association (Hirth/Nov15/914-793; Hirth/Oct13/6202; Hirth/Mar12/6085; Hirth/Oct07/6233), and Alzheimer’s Research UK (Hirth/ARUK/2012) to F.H.


  1. 1.
    Skeath JB, Thor S (2003) Genetic control of Drosophila nerve cord development. Curr Opin Neurobiol 13:8–15CrossRefGoogle Scholar
  2. 2.
    Homem CC, Repic M, Knoblich JA (2015) Proliferation control in neural stem and progenitor cells. Nat Rev Neurosci 16:647–659CrossRefGoogle Scholar
  3. 3.
    Urbach R, Technau GM (2004) Neuroblast formation and patterning during early brain development in Drosophila. BioEssays 26:739–751CrossRefGoogle Scholar
  4. 4.
    Hirth F, Reichert H (1999) Conserved genetic programs in insect and mammalian brain development. BioEssays 21:677–684CrossRefGoogle Scholar
  5. 5.
    Venken KJ, Simpson JH, Bellen HJ (2011) Genetic manipulation of genes and cells in the nervous system of the fruit fly. Neuron 72:202–230CrossRefGoogle Scholar
  6. 6.
    Shaw RE, Kottler B, Ludlow ZN, Buhl E, Kim D, Morais da Silva S, Miedzik A, Coum A, Hodge JJ, Hirth F, Sousa-Nunes R (2018) In vivo expansion of functionally integrated GABAergic interneurons by targeted increase in neural progenitors. EMBO J 37:e98163CrossRefGoogle Scholar
  7. 7.
    Diaper DC, Adachi Y, Lazarou L, Greenstein M, Simoes FA, Di Domenico A, Solomon DA, Lowe S, Alsubaie R, Cheng D, Buckley S, Humphrey DM, Shaw CE, Hirth F (2013) Drosophila TDP-43 dysfunction in glia and muscle cells cause cytological and behavioural phenotypes that characterize ALS and FTLD. Hum Mol Genet 22:3883–3893CrossRefGoogle Scholar
  8. 8.
    White KE, Humphrey DM, Hirth F (2010) The dopaminergic system in the aging brain of Drosophila. Front Neurosci 4:205CrossRefGoogle Scholar
  9. 9.
    Muqit MK, Feany MB (2002) Modelling neurodegenerative diseases in Drosophila: a fruitful approach? Nat Rev Neurosci 3:237–243CrossRefGoogle Scholar
  10. 10.
    Koizumi K, Higashida H, Yoo S et al (2007) RNA interference screen to identify genes required for Drosophila embryonic nervous system development. Proc Natl Acad Sci U S A 104:5626–5631CrossRefGoogle Scholar
  11. 11.
    Hirth F (2010) Drosophila melanogaster in the study of human neurodegeneration. CNS Neurol Disord Drug Targets 9:504–523CrossRefGoogle Scholar
  12. 12.
    Li T, Bellen HJ, Groves AK (2018) Using Drosophila to study mechanisms of hereditary hearing loss. Dis Model Mech 11:dmm031492CrossRefGoogle Scholar
  13. 13.
    Tsuji T, Higashida C, Yoshida Y et al (2011) Ect2, an ortholog of Drosophila’s pebble, negatively regulates neurite outgrowth in neuroblastoma × glioma hybrid NG108-15 cells. Cell Mol Neurobiol 31:663–668CrossRefGoogle Scholar
  14. 14.
    Venderova K, Kabbach G, Abdel-Messih E, Zhang Y, Parks RJ, Imai Y, Gehrke S, Ngsee J, Lavoie MJ, Slack RS, Rao Y, Zhang Z, Lu B, Haque ME, Park DS (2009) Leucine-Rich Repeat Kinase 2 interacts with Parkin, DJ-1 and PINK-1 in a Drosophila melanogaster model of Parkinson’s disease. Hum Mol Genet 18:4390–4404CrossRefGoogle Scholar
  15. 15.
    Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.
  16. 16.
    Patel N (1994) Imaging neuronal subsets and other cell types in whole mount Drosophila emrbyos and larvae using antibody probes. In: Goldstein LSB, Fryberg E (eds) Methods in cell biology, vol 44. Drosophila melanogaster: practical uses in cell biology. Academic, New York. For an amended and updated version, follow the link: Scholar
  17. 17.
    Ashburner M (1989) Drosophila: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  18. 18.
    Hoffman, G. (2008) Seeing is believing: use of antibodies in immunohistochemistry and in situ hybridization. In: Short course II of SfN’s 38 annual meeting: 15–19 November 2008. Society for Neuroscience, Washington, DCGoogle Scholar
  19. 19.
    Rothwell WF, Sullivan W (2000) Fluorescent analysis of Drosophila embryos. In: Sullivan W, Ashburner M, Hawley RS (eds) Drosophila protocols. Cold Spring Harbor Laboratory Press, New York, p 141Google Scholar
  20. 20.
    Bonaccorsi S, Giansanti MG, Cenci G, Gatti M (2012) Formaldehyde fixation of Drosophila testes. Cold Spring Harb Protoc:10.1101Google Scholar
  21. 21.
    Heimbeck G, Bugnon V, Gendre N, Häberlin C, Stocker RF (1999) Smell and taste perception in Drosophila melanogaster larva: toxin expression studies in chemosensory neurons. J Neurosci 19:6599–6609CrossRefGoogle Scholar
  22. 22.
    Stocker RF, Heimbeck G, Gendre N, de Belle JS (1997) Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of olfactory interneurons. J Neurobiol 32:443–456CrossRefGoogle Scholar
  23. 23.
    Hassell J, Hand AR (1974) Tissue fixation with diimidoesters as an alternative to aldehydes. I. Comparison of cross-linking and ultrastructure obtained with dimethylsuberimidate and glutaraldehyde. J Histochem Cytochem 22:223–229CrossRefGoogle Scholar
  24. 24.
    Wieschaus E, Nüsslein-Volhard C (1998) Looking at embryos. In: Roberts DB (ed) Drosophila, a practical approach. Oxford University Press, New York, p 205Google Scholar
  25. 25.
    Ripper D, Schwarz H, Stierhof YD (2008) Cryo-section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration. Biol Cell 100:109–123CrossRefGoogle Scholar
  26. 26.
    Rebay I, Fehon R (2000) Generating antibodies against Drosophila proteins. In: Sullivan W, Ashburner M, Hawley RS (eds) Drosophila protocols. Cold Spring Harbor Laboratory Press, New York, p 400Google Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and NeuroscienceKing’s College LondonLondonUK

Personalised recommendations