Skip to main content

In Situ Hybridization and Immunostaining of Xenopus Brain

  • Protocol
  • First Online:
Book cover Brain Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2047))

  • 2346 Accesses

Abstract

The dynamic expression pattern analysis provides the primary information of gene function. Differences of the RNA and/or protein location will provide valuable information for gene expression regulation. Generally, in situ hybridization (ISH) and immunohistochemistry (IHC) are two main techniques to visualize the locations of gene transcripts and protein products in situ, respectively. Here we describe the protocol for the whole brain dissection, the in situ hybridization, and the immunostaining of the developing Xenopus brain sections. Additionally, we point out the modification of in situ hybridization for microRNA expression detection.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
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. Harland RM (1991) In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol 36:685–695

    Article  CAS  Google Scholar 

  2. Lupo G, Liu Y, Qiu R, Chandraratna RA, Barsacchi G, He RQ, Harris WA (2005) Dorsoventral patterning of the Xenopus eye: a collaboration of Retinoid, Hedgehog and FGF receptor signaling. Development 132:1737–1748

    Article  CAS  Google Scholar 

  3. Liu Y, Lupo G, Marchitiello A, Gestri G, He RQ, Banfi S, Barsacchi G (2001) Expression of the Xvax2 gene demarcates presumptive ventral telencephalon and specific visual structures in Xenopus laevis. Mech Dev 100:115–118

    Article  CAS  Google Scholar 

  4. Lan L, Liu M, Liu Y, He R (2007) Expression and antibody preparation of POU transcription factor qBrn-1. Protein Pept Lett 14:7–11

    Article  CAS  Google Scholar 

  5. Lan L, Liu M, Liu Y, Zhang W, Xue J, Xue Z, He R (2006) Expression of qBrn-1, a new member of the POU gene family, in the early developing nervous system and embryonic kidney. Dev Dyn 235:1107–1114

    Article  CAS  Google Scholar 

  6. Saint-Jeannet JP (2017) Whole-mount in situ hybridization of Xenopus embryos. Cold Spring Harbor Protoc 2017:pdb prot097287. https://doi.org/10.1101/pdb.prot097287

    Article  Google Scholar 

  7. Decembrini S, Bressan D, Vignali R, Pitto L, Mariotti S, Rainaldi G, Wang X, Evangelista M, Barsacchi G, Cremisi F (2009) MicroRNAs couple cell fate and developmental timing in retina. Proc Natl Acad Sci U S A 106(50):21179–21184

    Article  CAS  Google Scholar 

  8. Liu K, Liu Y, Mo W, Qiu R, Wang X, Wu JY, He R (2011) MiR-124 regulates early neurogenesis in the optic vesicle and forebrain, targeting NeuroD1. Nucleic Acids Res 39:2869–2879

    Article  CAS  Google Scholar 

  9. Qiu R, Liu Y, Wu JY, Liu K, Mo W, He R (2009) Misexpression of miR-196a induces eye anomaly in Xenopus laevis. Brain Res Bull 79:26–31

    Article  CAS  Google Scholar 

  10. Qiu R, Liu K, Liu Y, Mo W, Flynt AS, Patton JG, Kar A, Wu JY, He R (2009) The role of miR-124a in early development of the Xenopus eye. Mech Dev 126:804–816

    Article  CAS  Google Scholar 

  11. Susaki EA, Tainaka K, Perrin D, Yukinaga H, Kuno A, Ueda HR (2015) Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protocols 10(11):1709–1727

    Article  CAS  Google Scholar 

  12. Sive HL, Grainger RM, Richard RM (2002) Early development of Xenopus laevis: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  13. Nieuwkoop PD, Faber J (1994) Normal table of Xenopus laevis (Daudin): a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. Garland, New York

    Google Scholar 

  14. Kloosterman WP, Wienholds E, de Bruijn E, Kauppinen S, Plasterk RH (2006) In situ detection of miRNAs in animal embryos using LNA modified oligonucleotide probes. Nat Methods 3:27–29

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Federico Cremisi for sharing the protocol of in situ hybridization with cryosections. This work was supported by the NSFC No.31271387.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ying Liu .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Liu, Kl., Wang, Xm., Li, Zl., Liu, Y., He, Rq. (2020). In Situ Hybridization and Immunostaining of Xenopus Brain. In: Sprecher, S. (eds) Brain Development. Methods in Molecular Biology, vol 2047. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9732-9_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9732-9_20

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9731-2

  • Online ISBN: 978-1-4939-9732-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics