I am using mouse liver tissue which is fixed in 4% PFA and embedded in OCT. However, autofluorescence signal is coming from the negative control samples, when Abcam protocol is used. Do you have any idea or any good protocol that could work with these conditions?
Hi Hilal,
Can you provide more details about your protocol?
Anyway, in this site there are some tips regarding autofluorescence:
http://www.ihcworld.com/autofluorescence.htm
Here you can also find some tips for get rid of autofluorescence in brain and liver:
http://www.ncbi.nlm.nih.gov/pubmed/11850446
Hope this helps,
cheers.
http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf
Here you can see some methods for autofluorescence quenching.
I once used 100 mM Glycine solution for 1 hour, but there are other ways as well.
Hi,
autofluorescence is usually only strong in the green channel (FITC, Alexa488), but not in the red (Rhodamine, Alexa 5XX etc.). If you do not do a co-stain, switching to red may solve the problem. Also, often you can distinguish signal from noise by comparing your stained section with secondary only control. If you have a decent signal, it will be considerably stronger than the autofluorescence. In that case, you just have to adjust the exposure time so the camera can see the signal, but not the noise.
Good luck!
Use protocols that have Sudan-Black blocking - that should block all your autofluorescence.
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