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.