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Light-Sheet Fluorescence Microscopy: Chemical Clearing and Labeling Protocols for Ultramicroscopy

Part of the Methods in Molecular Biology book series (MIMB,volume 1563)

Abstract

Light-sheet microscopy is an effective technique in neuroscience, developmental biology, and cancer research for visualizing and analyzing cellular networks and whole organs in three dimensions. Because this technique requires specimens to be translucent they commonly have to be cleared before microscopy inspection. Here, we provide 3DISCO based protocols for preparing cleared samples of immuno-stained neural networks, lectin-labeled vascular networks, and Methoxy-X04 labeled beta-amyloid plaques in mice. 3DISCO utilizes the lipophilic solvents tetrahydrofuran (THF) and dibenzylether (DBE) for dehydration and successive clearing. Crucial steps for obtaining transparent tissues and preserving the fragile endogenous GFP are the transcardial perfusion, as well as the proper implementation of the 3DISCO clearing process using peroxide free chemicals. We further provide a protocol for resin embedding of 3DISCO cleared specimens that allows long term archiving of samples for years with virtually no loss in signal quality.

Key words

  • Chemical clearing
  • Whole-mount immune-labeling
  • Vascular networks
  • Light-sheet microscopy
  • 3D reconstruction
  • 3DISCO
  • Peroxide elimination
  • Alzheimer disease

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Acknowledgment

We thank Massih Foroughipour for preparing the technical drawings of the custom-made specimen clamps.

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Correspondence to Nina Jährling .

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Jährling, N., Becker, K., Saghafi, S., Dodt, HU. (2017). Light-Sheet Fluorescence Microscopy: Chemical Clearing and Labeling Protocols for Ultramicroscopy. In: Markaki, Y., Harz, H. (eds) Light Microscopy. Methods in Molecular Biology, vol 1563. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6810-7_3

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  • DOI: https://doi.org/10.1007/978-1-4939-6810-7_3

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  • Publisher Name: Humana Press, New York, NY

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