Dear all,
We use different protocol to clear our organ like X-clarity, CUBIC, RapiClear etc… But we have some problem with the DNA Staining. I measure that we stained with DAPI +/- 600 µm. The majority of our samples are bigger than 6 mm.
Do you have some probes that work better than DAPI ?
I’ve seen good results using TO-PRO3 in the context of iDISCO+
iDISCO+ protocol: https://idisco.info/
Original paper (figure 4): https://www.cell.com/cell/fulltext/S0092-8674(14)01297-5
Full penetration into a half brain is easily acheivable.
Assuming your clearing is good, the problem with DAPI is that at lower wavelengths there is usually more scattering and autofluorescence. TO-PRO3 is in the far red region of the spectrum where there is less scattering and autofluroescence. Hence, better contrast deep in cleared samples.
Good luck.
Pablo
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Not sure if it will work in your setup but I use SiR-Hoescht to stain DNA. You can get it from Spirochrome. Alternatively, the Lukinavičius lab published an improved variant recently (also with fluorophores other than Hoescht). This version is fantastic.
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No personal experiences with labeling DNA in a cleared sample for lightsheet, but am a big fan of TO-PRO3 in general. Is it possible to express a resident histone protein (ie H2b) to mark the chromatin - this way, no worries about staining working well - a pain to make the model (mouse?), depending on what you’re doing, but once you have it, you have it and never have to stain again!
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