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Section Immunostaining for Protein Expression and Cell Proliferation Studies of Regenerating Fins

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Zebrafish

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2707))

Abstract

Adult zebrafish fins fully regenerate after resection, providing a highly accessible and remarkable vertebrate model of organ regeneration. Fin injury triggers wound epidermis formation and the dedifferentiation of injury-adjacent mature cells to establish an organized blastema of progenitor cells. Balanced cell proliferation and redifferentiation along with cell movements then progressively reestablish patterned tissues and restore the fin to its original size and shape. A mechanistic understanding of these coordinated cell behaviors and transitions requires direct knowledge of proteins in their physiological context, including expression, subcellular localization, and activity. Antibody-based staining of sectioned fins facilitates such high-resolution analyses of specific, native proteins. Therefore, such methods are mainstays of comprehensive, hypothesis-driven fin regeneration studies. However, section immunostaining requires labor-intensive, empirical optimization. Here, we present detailed, multistep procedures for antibody staining and co-detecting proliferating cells using paraffin and frozen fin sections. We include suggestions to avoid common pitfalls and to streamline the development of optimized, validated protocols for new and challenging antibodies.

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Acknowledgments

We thank Astra Henner for technical expertise and edits. The National Institute of General Medical Sciences provided research funding (1R01GM127761).

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Authors and Affiliations

  1. Institute of Molecular Biology, University of Oregon, Eugene, OR, USA

    Scott Stewart & Kryn Stankunas

  2. Department of Biology, University of Oregon, Eugene, OR, USA

    Kryn Stankunas

Authors
  1. Scott Stewart
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  2. Kryn Stankunas
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Corresponding authors

Correspondence to Scott Stewart or Kryn Stankunas .

Editor information

Editors and Affiliations

  1. Department of Pediatrics Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    James F. Amatruda

  2. Centre for Developmental Neurobiology, MRC Centre for NeuroDevelopmental Disorders, King’s College London, London, UK

    Corinne Houart

  3. Laboratory of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka, Japan

    Koichi Kawakami

  4. Duke Regeneration Center, Department of Cell Biology, Duke University Medical Center, Durham, NC, USA

    Kenneth D. Poss

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Stewart, S., Stankunas, K. (2024). Section Immunostaining for Protein Expression and Cell Proliferation Studies of Regenerating Fins. In: Amatruda, J.F., Houart, C., Kawakami, K., Poss, K.D. (eds) Zebrafish. Methods in Molecular Biology, vol 2707. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3401-1_16

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  • DOI: https://doi.org/10.1007/978-1-0716-3401-1_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3400-4

  • Online ISBN: 978-1-0716-3401-1

  • eBook Packages: Springer Protocols

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