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Immunofluorescent antibody staining of intact Drosophila larvae

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Abstract

Antibody staining is a vital technique for studying the development of many model organisms, including Drosophila. Reliable protocols have long been available for antibody staining of Drosophila whole-mount embryos and dissected larvae. By contrast, methods for staining whole larvae have rarely been reported, are unreliable, and fail to work on large third-instar larvae. This has become a major limitation to understanding the role of multitissue interactions such as neural circuit formation and cell metastasis. We have modified existing embryo protocols to develop a reliable method for antibody staining of whole Drosophila larvae of any developmental stage. The procedure consists of a bleach wash, enzymatic digestion, first fixation, 'cracking', second fixation, (optional) Proteinase K (Pro-K) or sonication treatment, antibody staining, clearing, and mounting. The method takes longer than typical antibody stains of dissected larval tissues—12 or 16 d, depending on the size of the larvae, compared with 2–3 d for embryos or dissected tissue stains—but time is saved by eliminating the need for larval dissections and by allowing hundreds of larvae to be batch-processed. The method also works well for staining embryos, even late-stage embryos with cuticles, allowing characterization from early embryogenesis to the end of larval development.

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Figure 1: Comparison of old and new staining pipelines.
Figure 2: Staining of newly hatched larvae (L0).
Figure 3: Staining of first-instar larvae (L1).
Figure 4: Staining of second-instar larvae (L2).
Figure 5: Staining of third-instar larvae (L3).
Figure 6: Staining of embryos.

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Change history

  • 20 April 2017

    In the version of this article initially published, two concentrations for Triton X-100 detergent were given in the blocking buffer ingredient list—0.3% is the correct concentration. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank E.S. Heckscher for her inspiration to begin this work, and B. Bowerman, K. Sugioka, and C.-H. Chuang for their help on the C. elegans protocols. This work was supported by the Howard Hughes Medical Institute, where C.Q.D. is an investigator.

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Authors and Affiliations

  1. Institute of Molecular Biology, Institute of Neuroscience, Howard Hughes Medical Institute, University of Oregon, Eugene, Oregon, USA

    Laurina Manning & Chris Q Doe

Authors
  1. Laurina Manning
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  2. Chris Q Doe
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Contributions

L.M. conceived and performed all experiments, collected all data, and wrote the first draft of the paper. C.Q.D. assembled the figures and provided comments on the text.

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Correspondence to Laurina Manning or Chris Q Doe.

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The authors declare no competing financial interests.

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Manning, L., Doe, C. Immunofluorescent antibody staining of intact Drosophila larvae. Nat Protoc 12, 1–14 (2017). https://doi.org/10.1038/nprot.2016.162

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