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Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin

Nature Protocols volume 6, pages 46–55 (2011)Cite this article

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Abstract

Histological techniques are critical for observing tissue and cellular morphology. In this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish embryos embedded in JB-4 plastic resin—a glycol methacrylate-based medium that results in excellent preservation of tissue morphology. In addition, we describe our procedures for staining plastic sections with toluidine blue or hematoxylin and eosin, and show how to couple these stains with whole-mount RNA in situ hybridization. We also describe how to maintain and visualize immunofluorescence and EGFP signals in JB-4 resin. The protocol we outline—from embryo preparation, embedding, sectioning and staining to visualization—can be accomplished in 3 d. Overall, we reinforce that plastic embedding can provide higher resolution of cellular details and is a valuable tool for cellular and morphological studies in zebrafish.

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Figure 1
Figure 2: Visual aid for embedding and sectioning zebrafish embryos in JB-4 resin.
Figure 3: Toluidine blue and H&E staining on JB-4 plastic sections.
Figure 4: RNA in situ hybridization, immunofluorescence and enhanced green fluorescence protein (EGFP) signals in JB-4 plastic resin.

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Acknowledgements

We thank K. Baker, K. Jaffe, N. Okabe, J. Rosen and J. Schottenfeld for providing samples and help with the protocol; H.J. Tsai for providing Tg(myl7:EGFP)twu34 strain; M. Pack for advice on immunofluorescence protocols; V. Grover for information on storage conditions for staining reagents; and members of the Burdine lab for critical reading of the manuscript. R.D.B. is the 44th Scholar of the Edward Mallinckrodt, Jr. Foundation, and funds from this award were used in support of this work. Funds from awards to R.D.B. from the New Jersey Commission on Cancer Research (04-2405-CCR-E0), the Polycystic Kidney Disease Foundation (no. 117b2r), and from the National Institute of Child Health and Human Development (R01-HD048584) were used in support of this work. J.S.-B. was supported by predoctoral award 05-2411-CCR-E0 from the New Jersey Commission on Cancer Research.

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Authors and Affiliations

  1. Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA

    Jessica Sullivan-Brown, Margaret E Bisher & Rebecca D Burdine

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  1. Jessica Sullivan-Brown
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Contributions

J.S.-B. and M.E.B. developed the protocol. R.D.B. supervised the work. J.S.-B., M.E.B. and R.D.B. wrote the manuscript.

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Correspondence to Rebecca D Burdine.

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The authors declare no competing financial interests.

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Sullivan-Brown, J., Bisher, M. & Burdine, R. Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin. Nat Protoc 6, 46–55 (2011). https://doi.org/10.1038/nprot.2010.165

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