General Research Topics > Neuroscience

brain - autofluorescence

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mosqito:
Hi. After few years of +/- working IF on brain free-floating section, I started to face a new problem. After cutting my PFA-perfused brains on vibratome, I see bizarre background signal, which is autofluorescent (seen in sections mounted on slides directly after cutting). The signal comes from neuronal cell bodies, looks like small deposits of smth, and is particularly annoying in the CA3 hippocampal region, but generally seen in the whole brain. I assume that it is caused by PFA. Washing in PBS-glycine does not help at all. I would like to ask whether anyone knows about alternative fixative (I've heard about the Z-fix), or the way of getting rid of it. Many thanks for your help.
Michal

niederhr:
I'm by no means an expert, but if you want to know if your autofluorescence is caused by the PFA (or the aldehydes resulting from the PFA), can you try comparing to a section fixed with something like Histochoice or another aldehyde-free alternative?

If you're stuck with PFA, supposedly sodium borohydride will help, there are several different concentrations and incubation times out there, perhaps someone else can give specifics on that. I've also heard that using a Tris buffer helps, you may want to try your glycine block in TBS? Also, an  ammonium chloride wash is supposed to help. I can't vouch for their effectiveness personally. All I can really suggest is to Google or Pubmed search these methods and see if someone else has tried them in a situation similar to yours.

If the autofluorescence is NOT aldehyde-associated, it could be any one or more of many things. You might want to read the following PDF for some ideas:
http://www.uhnresearch.ca/facilities/wcif/PDF/Autofluorescence.pdf

Good luck!

mosqito:
Thank you for the response. Actually, I performed quite thourough search before posting the call for help, but still I decided to ask before checking everything. Meanwhile, I did an obvious thing, and checked the non-perfused frozen sections. The result is: same thing, CA3 is shining, although not so strong. Since I am trying to colocalize the protein with cellular markers, I really need a clear background. So, I decided to concentrate on DAB staining until new ideas appear (ANYONE?  :)).

KevinCao:
do bleach with light? sorry how would I accomplish that? Is it a valid tactic to use uv or some sort of light to completely irradiate all the samples before staining?

vgoose:
Mosqito - I have seen the same thing in my hippocampal sections fixed with PFA.  It almost looked like real labeling at first until I did some control exps that showed it is in fact auto f.  I typically only see it in the green channel and if I am using a robust antibody in that channel like GFAP-488 conjugated, it does not seem to be a big problem.  Only when you have low signal-to-noise does this auto f. rear its ugly head.  I have read that soaking your sections BEFORE you block with ammonium chloride can help... I have since switched to cryosections and shorter fixation times and it has seemed to go away. I was previously perfusing, post-fixing overnight and making vibratome sections.

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