I used Fluorophore Conjugated primary antibody (Alexa-488, 555 and 647). Tissue FFPE samples were imaged before I stainded antibody and I found background fluorescence.
So, my question is "what is the best of reduce autofluorescence in FFPE tissue using between sudan black b , TrueVIEW® and TrueBlack® ?"
And If you any methods for reduce autofluorescence please tell me.
Thank you
Blocking for some more time may help you. Additionally, use higher dilution for the antibody.
I hope, probably it should help.
What type of tissue do you use and how strong is the autofluorescence? FFPE fixed tissue tends to exhibit background fluorescence, but as long as the antibody signal is strong enough to be clearly distinguishable, this doesn't pose a problem.
I don't have experience with TrueVIEW and TrueBlack, but I did use the (probably cheaper) Sudan Black. The method worked fine in organotypic brain slice culture (FFPE), which had very high background fluorescence. However, it could not improve the signal-to-noise ratio in normal mouse brain tissue (FFPE), because the counter-staining with Sudan Black also quenches the signal of the actual staining. This effect might be decreased in the commercial products.
In case you want to try Sudan Black, this is how I used it:
- 0.1% SB dissolved in 70% EtOH
- filter & transfer to dripping bottle (as supplied in various staining kits)
- use 1-2 drops per section
I tested several condition. If counter-staining before or after primary antibody staining works better was inconclusive, but in your case I would assume doing it before antibody incubation is preferable.
Concerning the incubation time, I would recommend you to optimize it for your type of tissue & antibody.
- Wash thoroughly after the counter-staining before moving on to the next step.
Best wishes!
In addition to the above answer (by Suckert Theresa ), I have used Eriochrome black T, it works really well for skin and oral FFPE samples.
you may check this link for a related paper:
Article Characterizing and Diminishing Autofluorescence in Formalin-...
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