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Chemical clearing and dehydration of GFP expressing mouse brains.

Becker K, Jährling N, Saghafi S, Weiler R, Dodt HU - PLoS ONE (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioelectronics, Vienna University of Technology, FKE, Vienna, Austria. klaus.becker@meduniwien.ac.at

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.

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Related in: MedlinePlus

UM-reconstructions of isolated hippocampi of a thy-1 EGFP-M mouse.DBE clearing provides improved fluorescence and visibility of details. A: left hippocampus dehydrated with THF and cleared with BABB. B: corresponding right hippocampus dehydrated with THF and cleared with DBE. A1–B1: Olympus objective XLFluar 4×, N.A. 0.28, scale bar 100 µm. (A2–B2) 4× Olympus objective XLFluar 4×, N.A. 0.28, scale bar 20 µm. A3–B3: 4× Olympus objective XLFluar 4×, N.A. 0.28, scale bar 10 µm. CA1: cornu ammonis region one. gl: granular cell layer py: pyramidal cells.
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pone-0033916-g003: UM-reconstructions of isolated hippocampi of a thy-1 EGFP-M mouse.DBE clearing provides improved fluorescence and visibility of details. A: left hippocampus dehydrated with THF and cleared with BABB. B: corresponding right hippocampus dehydrated with THF and cleared with DBE. A1–B1: Olympus objective XLFluar 4×, N.A. 0.28, scale bar 100 µm. (A2–B2) 4× Olympus objective XLFluar 4×, N.A. 0.28, scale bar 20 µm. A3–B3: 4× Olympus objective XLFluar 4×, N.A. 0.28, scale bar 10 µm. CA1: cornu ammonis region one. gl: granular cell layer py: pyramidal cells.

Mentions: We further prepared isolated mouse hippocampi [6], dehydrated them with THF, and cleared them with BABB (left hippocampus), or DBE (corresponding right hippocampus). As in the brain hemispheres, UM-reconstructions [6] verify the strikingly improved fluorescence signal intensity obtained by DBE-clearing (Figure 3).


Chemical clearing and dehydration of GFP expressing mouse brains.

Becker K, Jährling N, Saghafi S, Weiler R, Dodt HU - PLoS ONE (2012)

UM-reconstructions of isolated hippocampi of a thy-1 EGFP-M mouse.DBE clearing provides improved fluorescence and visibility of details. A: left hippocampus dehydrated with THF and cleared with BABB. B: corresponding right hippocampus dehydrated with THF and cleared with DBE. A1–B1: Olympus objective XLFluar 4×, N.A. 0.28, scale bar 100 µm. (A2–B2) 4× Olympus objective XLFluar 4×, N.A. 0.28, scale bar 20 µm. A3–B3: 4× Olympus objective XLFluar 4×, N.A. 0.28, scale bar 10 µm. CA1: cornu ammonis region one. gl: granular cell layer py: pyramidal cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316521&req=5

pone-0033916-g003: UM-reconstructions of isolated hippocampi of a thy-1 EGFP-M mouse.DBE clearing provides improved fluorescence and visibility of details. A: left hippocampus dehydrated with THF and cleared with BABB. B: corresponding right hippocampus dehydrated with THF and cleared with DBE. A1–B1: Olympus objective XLFluar 4×, N.A. 0.28, scale bar 100 µm. (A2–B2) 4× Olympus objective XLFluar 4×, N.A. 0.28, scale bar 20 µm. A3–B3: 4× Olympus objective XLFluar 4×, N.A. 0.28, scale bar 10 µm. CA1: cornu ammonis region one. gl: granular cell layer py: pyramidal cells.
Mentions: We further prepared isolated mouse hippocampi [6], dehydrated them with THF, and cleared them with BABB (left hippocampus), or DBE (corresponding right hippocampus). As in the brain hemispheres, UM-reconstructions [6] verify the strikingly improved fluorescence signal intensity obtained by DBE-clearing (Figure 3).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioelectronics, Vienna University of Technology, FKE, Vienna, Austria. klaus.becker@meduniwien.ac.at

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.

Show MeSH
Related in: MedlinePlus