Advertisement

Xenopus pp 233-241 | Cite as

X-FaCT: Xenopus-Fast Clearing Technique

  • Pierre Affaticati
  • Sébastien Le Mével
  • Arnim Jenett
  • Laurie Rivière
  • Elodie Machado
  • Bilal B. Mughal
  • Jean-Baptiste FiniEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1865)

Abstract

Accessibility and imaging of cell compartments in big specimens are crucial for cellular biological research but also a matter of contention. Confocal imaging and tissue clearing on whole organs allow for 3D imaging of cellular structures after being subjected to in-toto immunohistochemistry. Lately, the passive CLARITY technique (PACT) has been adapted to clear and immunolabel large specimens or individual organs of several aquatic species. We recently demonstrated tissue clearing on one-week old tadpole brain (Fini et al., Sci Rep 7:43786, 2017). We here describe a further simplified version with clearing of small tissue samples (thickness inferior to 500 μm)) carried out by immersion in a fructose-based high-refractive index solution (fbHRI). By refining steps of the protocol, we were able to reduce the overall procedure time by two thirds. This offers the advantages of reducing the time of experimentation to a week and minimizes procedure-induced tissue deformations. This protocol can be easily adapted to be performed on thick section. We present an example of immunohistochemistry performed on NF45 Xenopus laevis brains with anti-pH 3 (phosphorylated histone H3) antibody used to stain chromatin condensation commonly associated with proliferation.

Key words

Tissue clearing Fructose-based high-refractive index solution 3D imaging 

Supplementary material

Movie 1

Dissection of brain NF45. Tadpole after fixation is maintained with a pair of forceps. Procedure relies on removing the skin and then to use fine tool under the brain and to cut the spinal cord (MP4 7224 kb)

Movie 2

Bottom to top (in dorsal view) video of confocal planes of a NF45 control brain stained with DiD (gray), immunolabeled for pH3 (green) (MP4 8241 kb)

Movie 3

Bottom to top video of confocal planes of a NF45 T3 5nM treated brain stained with DiD (gray), immunolabeled for pH3 (green) (MP4 8414 kb)

Movie 4

Rotation movie on sagittal axis of a control 3D reconstructed brain immunolabeled for pH3 (green) (MP4 950 kb)

Movie 5

Rotation movie on sagittal axis of a T3 treated, 3D reconstructed brain immunolabeled for pH3 (green) (MP4 1481 kb)

References

  1. 1.
    Chung K, Wallace J, Kim SY, Kalyanasundaram S, Andalman AS, Davidson TJ, Mirzabekov JJ, Zalocusky KA, Mattis J, Denisin AK, Pak S, Bernstein H, Ramakrishnan C, Grosenick L, Gradinaru V, Deisseroth K (2013) Structural and molecular interrogation of intact biological systems. Nature 497(7449):332–337.  https://doi.org/10.1038/nature12107CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Tomer R, Ye L, Hsueh B, Deisseroth K (2014) Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat Protoc 9(7):1682–1697.  https://doi.org/10.1038/nprot.2014.123CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Treweek JB, Chan KY, Flytzanis NC, Yang B, Deverman BE, Greenbaum A, Lignell A, Xiao C, Cai L, Ladinsky MS, Bjorkman PJ, Fowlkes CC, Gradinaru V (2015) Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. Nat Protoc 10(11):1860–1896.  https://doi.org/10.1038/nprot.2015.122CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Yang B, Treweek JB, Kulkarni RP, Deverman BE, Chen CK, Lubeck E, Shah S, Cai L, Gradinaru V (2014) Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158(4):945–958.  https://doi.org/10.1016/j.cell.2014.07.017CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Dambroise E, Simion M, Bourquard T, Bouffard S, Rizzi B, Jaszczyszyn Y, Bourge M, Affaticati P, Heuze A, Jouralet J, Edouard J, Brown S, Thermes C, Poupon A, Reiter E, Sohm F, Bourrat F, Joly JS (2017) Postembryonic fish brain proliferation zones exhibit neuroepithelial-type gene expression profile. Stem Cells 35(6):1505–1518.  https://doi.org/10.1002/stem.2588CrossRefPubMedGoogle Scholar
  6. 6.
    Affaticati P, Simion M, De Job E, Rivière L, Hermel J, Machado E, Joly J, Jenett A (2017) zPACT: tissue clearing and immunohistochemistry on juvenile zebrafish brain. Bio-Protocol 7(23):e2636.  https://doi.org/10.21769/BioProtoc.2636CrossRefGoogle Scholar
  7. 7.
    Fini JB, Mughal BB, Le Mevel S, Leemans M, Lettmann M, Spirhanzlova P, Affaticati P, Jenett A, Demeneix BA (2017) Human amniotic fluid contaminants alter thyroid hormone signalling and early brain development in Xenopus embryos. Sci Rep 7:43786.  https://doi.org/10.1038/srep43786CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682.  https://doi.org/10.1038/nmeth.2019CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Pierre Affaticati
    • 1
  • Sébastien Le Mével
    • 2
  • Arnim Jenett
    • 1
  • Laurie Rivière
    • 1
  • Elodie Machado
    • 1
  • Bilal B. Mughal
    • 2
  • Jean-Baptiste Fini
    • 2
    Email author
  1. 1.Tefor Core Facility, Paris-Saclay Institute of NeuroscienceCNRS, Université Paris-SaclayGif-sur-YvetteFrance
  2. 2.Evolution des Régulations Endocriniennes, Département « Adaptation du Vivant »UMR 7221 MNHN/CNRS, Sorbonne UniversitésParisFrance

Personalised recommendations