Skip to main content
SpringerLink
Log in

Fixation/Permeabilization Procedure for mRNA In Situ Hybridization of Zebrafish Whole-Mount Oocytes, Embryos, and Larvae

  • Protocol
  • First Online:
In Situ Hybridization Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1211))

Abstract

A new procedure for improved in situ hybridization of zebrafish whole-mount oocytes, embryos, and early larvae is described. The procedure relies on the simultaneous fixation/permeabilization of samples using formaldehyde as fixative and short C-chain aliphatic carboxylic acids, particularly glacial acetic acid, as permeabilizers. As compared with in situ hybridization performed with routine methods, our procedure is simpler and provides better structural preservation of cells and tissues, equivalent mRNA signals, and similar results in embryos of different developmental stages. It is hypothesized that during aldehyde fixation short C-chain aliphatic carboxylic acids modulate the rate of formation and/or destruction of methylene bridges established between cell proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Fernández J, Fuentes R (2013) Fixation/permeabilization: new alternative procedure for immunofluorescence and mRNA in situ hybridization of vertebrate and invertebrate embryos. Dev Dyn 242:503–517

    Article  PubMed  Google Scholar 

  2. Hayat MA (2002) Microscopy, immunohistochemistry and antigen retrieval methods. For light and electron microscopy. Kluwer Academic Publishers, New York

    Google Scholar 

  3. Kiernan JA (2000) Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: what they are and what they do. Microsc Today 1:8–12

    Google Scholar 

  4. Puchtler H, Meloan SN (1985) On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochemistry 82:201–204

    Article  PubMed  CAS  Google Scholar 

  5. Sutherland BW, Toews J, Kast J (2008) Utility of formaldehyde cross-linking and mass spectroscopy in the study of protein-protein interactions. J Mass Spectrom 43:699–715

    Article  PubMed  CAS  Google Scholar 

  6. Dedon PC, Soults JA, Allis D et al (1991) Formaldehyde cross-linking and immunoprecipitation demonstrate developmental changes in H1 association with transcriptionally active genes. Mol Cell Biol 11:1729–1733

    PubMed  CAS  PubMed Central  Google Scholar 

  7. Orlando V, Strutt H, Paro R (1997) Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods 11:205–214

    Article  PubMed  CAS  Google Scholar 

  8. Spencer VA, Davie JR (2002) Isolation of protein cross-linked to DNA by formaldehyde. In: Walker JM (ed) The protein protocols handbook. Humana Press Inc., Totowa, NJ, pp 753–757

    Chapter  Google Scholar 

  9. Schmiedeberg L, Skene P, Deaton A et al (2009) A temporal threshold for formaldehyde cross-linking and fixation. PLoS One 4:1–5

    Article  Google Scholar 

  10. Fernández J, Olea N, Tellez V et al (1990) Structure and development of the egg of the glossiphoniid leech Theromyzon rude: reorganization of the fertilized egg during completion of the first meiotic division. Dev Biol 137:142–154

    Article  PubMed  Google Scholar 

  11. Fernández J, Olea N, Tellez V (1994) Formation of the male pronucleus, organization of the first interphase monaster and establishment of the perinuclear plasm domain in the egg of the glossiphoniid leech Theromyzon rude. Dev Biol 164:111–122

    Article  PubMed  Google Scholar 

  12. Fernández J, Olea N, Ubilla A et al (1998) Formation of polar cytoplasmic domains (teloplasms) in the leech egg is a three-step segregation process. Int J Dev Biol 42:149–162

    PubMed  Google Scholar 

  13. Fernández J, Olea N (1995) Formation of the female pronucleus and reorganization and disassembly of the first interphase cytoskeleton in the egg of the glossiphoniid leech Theromyzon rude. Dev Biol 171:541–553

    Article  PubMed  Google Scholar 

  14. Cantillana V, Urrutia M, Ubilla A et al (2000) The complex dynamic network of microtubule and microfilament cytasters of the leech zygote. Dev Biol 228:136–149

    Article  PubMed  CAS  Google Scholar 

  15. Gard DL (1991) Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: a study by confocal immunofluorescence microscopy. Dev Biol 143:346–362

    Article  PubMed  CAS  Google Scholar 

  16. Schroeder MM, Gard DL (1992) Organization and regulation of cortical microtubules during the first cell cycle of Xenopus eggs. Development 11:699–709

    Google Scholar 

  17. Pelegri F, Knaut H, Maischein HM et al (1999) A mutation in the zebrafish maternal-effect gene nebel affects furrow formation and vasa RNA localization. Curr Biol 9:1431–1440

    Article  PubMed  CAS  Google Scholar 

  18. Hammati-Brivenlou A, Harland RM (1989) Expression of an engrailed related protein is induced in the anterior neural ectoderm of early Xenopus embryos. Development 106:611–617

    Google Scholar 

  19. Harland RM (1991) In situ hybridization: an improved whole-mount method for Xenopus embryos. In: Kay BK, Peng HB (eds) Methods in cell biology. Academic Press Inc., San Diego, pp 685–695

    Google Scholar 

  20. Schulte-Merker S, Ho RK, Herrmann BG et al (1992) The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116:1021–1032

    PubMed  CAS  Google Scholar 

  21. Stachel S, Grunwald DJ, Myers PZ (1993) Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish. Development 117:1261–1274

    PubMed  CAS  Google Scholar 

  22. Strähle U, Jesuthasan S (1993) Ultraviolet irradiation impairs epiboly in zebrafish embryos: evidence for microtubule dependent mechanism of epiboly. Development 119:909–919

    PubMed  Google Scholar 

  23. Strähle U, Blader P, Henrique D et al (1993) Axial, a zebrafish gene expressed along the developing body axis, shows altered expression in cyclops mutant embryos. Genes Dev 7:1436–1446

    Article  PubMed  Google Scholar 

  24. Henrique D, Adam J, Myat A et al (1995) Expression of a Delta homologue in prospective neurons in the chick. Nature 375:787–790

    Article  PubMed  CAS  Google Scholar 

  25. Allende M, Amsterdam A, Becker T et al (1996) Insertional mutagenesis in zebrafish identifies two novel genes, pescadillo and dead eye, essential for embryonic development. Genes Dev 10:3141–3155

    Article  PubMed  CAS  Google Scholar 

  26. Jesuthasan S (1998) Furrow-associated microtubule arrays are required for the cohesion of zebrafish blastomeres following cytokinesis. J Cell Sci 111:3695–3703

    PubMed  CAS  Google Scholar 

  27. Leung CF, Webb SE, Miller AL (2000) On the mechanism of ooplasmic segregation in single-cell zebrafish embryos. Dev Growth Differ 42:29–40

    Article  PubMed  CAS  Google Scholar 

  28. Streit A, Stern CD (2001) Combined whole-mount in situ hybridization and immunohistochemistry in avian embryos. Methods 23:339–344

    Article  PubMed  CAS  Google Scholar 

  29. Kang D, Pillon M, Weisblat DA (2002) Maternal and zygotic expression of a nanos-class gene in the leech Helobdella robusta: primordial germ cells arise from segmental mesoderm. Dev Biol 245:28–41

    Article  PubMed  CAS  Google Scholar 

  30. Dekens MPS, Pelegri F, Maischein HM et al (2003) The maternal-effect gene futile cycle is essential for pronuclear congression and mitotic spindle assembly in the zebrafish zygote. Development 130:3907–3916

    Article  PubMed  CAS  Google Scholar 

  31. Thisse C, Thisse B (2008) High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc 3:59–69

    Article  PubMed  CAS  Google Scholar 

  32. Renshaw S (2007) Immunohistochemistry: methods express. Scion Publishing Ltd, Bloxham, UK

    Google Scholar 

  33. Fernández J (1980) Embryonic development of the glossiphoniid leech Theromyzon rude: characterization of developmental stages. Dev Biol 78:407–434

    Article  PubMed  Google Scholar 

  34. Gupta T, Fl M, Ferriola D, Mackiewicz K, Dapprich J, Monos D, Mullins MC (2010). Microtubule actin crosslinking factor 1 regulates the Balbiani body and animal-vegetal polarity of the zebrafish oocyte. PLoS Genet 6:e1001073

    Google Scholar 

  35. Selman K, Wallace RA, Sarka A et al (1993) Stages of oocyte development in the zebrafish, Brachydanio rerio. J Morphol 218:203–224

    Article  Google Scholar 

  36. Kimmel CB, Ballard WW, Kimmel SR et al (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203:253–310

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Drs. M.C. Mullins, F. Pelegri, V. Gallardo, A. Reyes, and L. Valdivia for donation of cDNA-inserted plasmids or riboprobes and V. Guzman for technical assistance. Dr. M.C. Mullins kindly provided her laboratory facilities to perform some experiments. Financed by the University of Chile.

Author information

Authors and Affiliations

  1. Department of Cell and Developmental Biology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, 19104, USA

    Ricardo Fuentes

  2. Department of Biology, Faculty of Sciences, University of Chile, Santiago, 7800003, Chile

    Juan Fernández

Authors
  1. Ricardo Fuentes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Juan Fernández
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Juan Fernández .

Editor information

Editors and Affiliations

  1. Molecular Histology, Bioneer A/S, Horsholm, Denmark

    Boye Schnack Nielsen

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Fuentes, R., Fernández, J. (2014). Fixation/Permeabilization Procedure for mRNA In Situ Hybridization of Zebrafish Whole-Mount Oocytes, Embryos, and Larvae. In: Nielsen, B. (eds) In Situ Hybridization Protocols. Methods in Molecular Biology, vol 1211. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1459-3_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1459-3_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1458-6

  • Online ISBN: 978-1-4939-1459-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation