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PLoS One. 2015 May 20;10(5):e0124650. doi: 10.1371/journal.pone.0124650. eCollection 2015.

Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains.

Author information

1
Max Planck Institute for Medical Research, Heidelberg, Germany.
2
German Cancer Research Center (DKFZ), Heidelberg, Germany.
3
Center for Molecular Biology (ZMBH), University of Heidelberg, Heidelberg, Germany.

Abstract

In order to observe and quantify long-range neuronal connections in intact mouse brain by light microscopy, it is first necessary to clear the brain, thus suppressing refractive-index variations. Here we describe a method that clears the brain and preserves the signal from proteinaceous fluorophores using a pH-adjusted non-aqueous index-matching medium. Successful clearing is enabled through the use of either 1-propanol or tert-butanol during dehydration whilst maintaining a basic pH. We show that high-resolution fluorescence imaging of entire, structurally intact juvenile and adult mouse brains is possible at subcellular resolution, even following many months in clearing solution. We also show that axonal long-range projections that are EGFP-labelled by modified Rabies virus can be imaged throughout the brain using a purpose-built light-sheet fluorescence microscope. To demonstrate the viability of the technique, we determined a detailed map of the monosynaptic projections onto a target cell population in the lateral entorhinal cortex. This example demonstrates that our method permits the quantification of whole-brain connectivity patterns at the subcellular level in the uncut brain.

PMID:
25993380
PMCID:
PMC4439039
DOI:
10.1371/journal.pone.0124650
[Indexed for MEDLINE]
Free PMC Article

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