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.
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1 Electronic Supplementary Material
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)
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)
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)
Rotation movie on sagittal axis of a control 3D reconstructed brain immunolabeled for pH3 (green) (MP4 950 kb)
Rotation movie on sagittal axis of a T3 treated, 3D reconstructed brain immunolabeled for pH3 (green) (MP4 1481 kb)
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Affaticati, P. et al. (2018). X-FaCT: Xenopus-Fast Clearing Technique. In: Vleminckx, K. (eds) Xenopus. Methods in Molecular Biology, vol 1865. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8784-9_16
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DOI: https://doi.org/10.1007/978-1-4939-8784-9_16
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