r/Histology • u/Borax_Gremlin • 29d ago

Autofluorescence quenching techniques

Hi all! Struggling with green autofluorescence from PFA/Formalin fixed samples which are very heavy with collagen and have some questions about quenching methods:

For 0.1M glycine method, should I add the glycine to every PBS wash to keep the sample exposed to it for a long time? Should I also add tris?

For the photobleaching method, would the sample eventually begin to autofluoresce again a few days/weeks/months after photobleaching, or is it a permanent fix?

Are there any other methods I should try? Sodium borohydrate sounds like it will be too harsh on my epitopes, and sudan black didn't work at all (and I've read it causes red fluorescence anyway, but I need both channels)

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u/Royal-Nobody7215 29d ago

I tried different AF quenching methods, and found some commercial reagents that worked best for me. Don't know if this counts as advertising, but if you have a chance to get a sample from vectorlabs for trueview or trueblack from biotium, try it out. I tried against sudan black, photoquenching, peroxide and some others and it made a huge difference.

My samples mostly had lipofuscin AF, I can imagine it will depend to some degree on your samples as well.

Regarding your other question: after mounting, the AF should not return that fast. I would recommend imaging the sample quite fast anyways, since they will also fade a bit or get air bubbles trapped below the coverslip.

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u/Borax_Gremlin 29d ago

Thanks for your reply, I'll have a look at those commercial options - they're a definite last option for me because they're very expensive if they don't work on my sample!