Hi, I have GFP-expressing cells growing in a scaffold that is highly autofluorescent in the green band. Ideally i would like to fix, mount and visualise the GFP within the cells. I know that treatment with NaBH4 reduces autofluorescence for IHC or other applications, but does anyone know if this will also affect the fluorescence of the GFP I hope to detect?
Thanks :)
Hi there Luke,
Apparently, the chemical mechanism by which NaBH4 decreases auto-fluorescence is a reduction from a chemical point of view, same thing as for NH4Cl.
"NaBH4 is known to neutralize Schiffs bases through reduction of amine-aldehyde compounds into non-fluorescent salts."
https://pubmed.ncbi.nlm.nih.gov/9765122/
If that is the case, it shouldn't affect the remaining fluorescence of your GFP after fixation, although after PFA treatment, I wouldn't expect the latter to be optimal in any case.
Hope this helps,
Cheers,
J
If quenching the background does not work well, there is also the option to label/enhance the GFP signal. The easiest way is probably using a GFP booster e.g. conjugated to a red Alexa Fluor.
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