Dear Matthew,

I experience the same problem for colonic FFPE sections. If your antibodies display strong staining, probably the best option is to quench autofluorescence using Sudan Black (or the commercial quencher from Dianova) for varying times after retrieval and before antibody incubation. This does not completely eliminate the autofluorescence, but helps to distinguish the antibody-specific signal by increasing the signal-to-noise ratio.

I agree with Matthias on the Sudan Black B, though I've read that applying it before antibody incubation may prevent binding. It may be best when done after secondary antibody incubations. An alternative post-treatment is copper sulfate in ammonium acetate. Pre-treatment of tissue sections with sodium borohydride in distilled water may also reduce formalin-fixation autofluorescence.

We have been fighting with the problem of autofluorescence in formalin-fixed sections for quite a while and finally discovered that one should simply cut the time of fixing. The reagents are now so pure that to fix a sample up to 1.5 cm thick one does not need 12 -24 hours of incubation in formalin as prescribed in classical protocols. 1-2 h Room Temperature 4% buffered formalin proved to be enough for all our stains and applications. It allows one to double the profit as it speeds up the process and eliminates the problem of autofluorescence. The finding was tested on multiple murine and human (muscle biopsy) tissue samples. Three years of experience - great confocal images - highly recommended.

Thank you, Elena, for your contribution to Matthew's question.

Would you please refer us (or me) to a particular publication you used such protocol? I always wanted to fix for less time, but I haven't got a lead yet. I work with both full brain and slices which I fix for 24 hrs and 30 minutes, respectively.

Thank you!