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Hello Tingting,

Most often it is the dehydration step that causes the loss of fluorescence.
FPs require water molecules to maintain their confirmation, once these are
removed, the chromophore falls apart and you lose fluorescence. Methanol is
the worst for this, the emission will be lost almost immediately.
Ethanol is a little better and THF will give you a day or two of weak
fluorescence. The final clearing solution (DBE, ECi, etc.) also plays a
role, but the dehydration step does the most damage.

The most recent solvent-based clearing protocols have found some tricks to
better protect the FPs and preserve fluorescence for weeks or months. These
include:

1) dehydration with 1-propanol or tert-butanol (or a mixture of both)
2) Making sure all solutions have a basic pH (~9.0)
3) Keeping the sample at 4C during clearing
4) Supplementing the solutions with Quadrol or Polyethylene Glycol

For more info see:
•uDISCO –Pan et al., Nature Methods, 2016
•a-uDISCO – Liet al., Frontiers in Neuroanatomy, 2018
•PEGASOS– Cell Research, 2018

-Doug


On Mon, May 24, 2021 at 2:22 AM Gu, Tingting <[log in to unmask]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear all,
>
> We tried Ethanol/ECi-based tissue clearing on the whole mouse brain with
> endogenous TdTomato fluorescent signal. After the clearing, no
> TdTomato-specific signal left, but high autofluorescent background in RFP
> range. ECi cleared brain has some yellow color but it is clear.
>
> Do anyone have experience of using ECi-based tissue clearing method to
> clear mouse brain with endogenous TdTomato signals? Or any recommendations
> on which clearing methods can preserve endogenous TdTomato signals. In
> addition, how to reduce autofluorescent background?
>
> Any suggestions would be greatly appreciated.
> Best,
> Tingting
>