Chemical Clearing and Dehydration of GFP Expressing Mouse Brains
Open Access
- 30 March 2012
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 7 (3), e33916
- https://doi.org/10.1371/journal.pone.0033916
Abstract
Generally, chemical tissue clearing is performed by a solution consisting of two parts benzyl benzoate and one part benzyl alcohol. However, prolonged exposure to this mixture markedly reduces the fluorescence of GFP expressing specimens, so that one has to compromise between clearing quality and fluorescence preservation. This can be a severe drawback when working with specimens exhibiting low GFP expression rates. Thus, we screened for a substitute and found that dibenzyl ether (phenylmethoxymethylbenzene, CAS 103-50-4) can be applied as a more GFP-friendly clearing medium. Clearing with dibenzyl ether provides improved tissue transparency and strikingly improved fluorescence intensity in GFP expressing mouse brains and other samples as mouse spinal cords, or embryos. Chemical clearing, staining, and embedding of biological samples mostly requires careful foregoing tissue dehydration. The commonly applied tissue dehydration medium is ethanol, which also can markedly impair GFP fluorescence. Screening for a substitute also for ethanol we found that tetrahydrofuran (CAS 109-99-9) is a more GFP-friendly dehydration medium than ethanol, providing better tissue transparency obtained by successive clearing. Combined, tetrahydrofuran and dibenzyl ether allow dehydration and chemical clearing of even delicate samples for UM, confocal microscopy, and other microscopy techniques.This publication has 13 references indexed in Scilit:
- Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injuryNature Medicine, 2011
- Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brainNature Neuroscience, 2011
- Oxyradical-induced GFP damage and loss of fluorescenceInternational Journal of Biological Macromolecules, 2008
- Ultramicroscopy: 3D reconstruction of large microscopical specimensJournal of Biophotonics, 2008
- Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brainNature Methods, 2007
- Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFPNeuron, 2000
- THE GREEN FLUORESCENT PROTEINAnnual Review of Biochemistry, 1998
- Safety evaluation of dibenzyl etherFood and Chemical Toxicology, 1992
- New Reflected-Light Microscope for Viewing Unstained Brain and Ganglion CellsScience, 1967
- Use of Tetrahydrofuran for Routine and Rapid Dehydration and ClearingArchives of Dermatology, 1960