We are using Lumafluor red retrobeads to label DRG neurones, which works brilliantly when recording from neurones in culture.
However, for immunohistochemical studies we observe high background fluorescence, such that the "bead-like" pattern produces higher fluorescence in the DAPI channel than when excited at 540nm, which by the excitation/emission spectra of the beads (https://lumafluor.com/Information.php) should not be the case, e.g. the bead-like things are autofluorescent, lipfuscin is 1 possible candidate I suppose.
For tissue preparation, animals are killed, DRG fixed in 4% PFA (pH roughly 7.4) for 20 minutes, cryopresevered in sucrose overnight and then sectioned on a cryostat (12µm sections).
Because of the difference we observe between fresh cultures and fixed sections, my guess is that something occurs during fixation and I would grateful for any tips and advice with regards to other fixing methods etc.
Thanks in advance!
Fixative induced fluorescence caused by aldehyde fixatives is predominantly in the green/yellow spectral range. In basic terms it's due to the change in strucutre caused by the cross linking and reaction of the fixative. Things with lots of chemical bonds such as C=C also produce a lot of autofluorescence.
There is a good guide here to different causes and cures: http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf
My advice would be to try a different aldehyde free fixative like methanol or reduced aldehyde such as Zamboni's or PLP. People generally find these result in less autofluorescence.
An alternative startegy is to pre-photobelach your samples before staining. This works but has mixed effects on antigens.
Hi Ewan,
As far as I know, neuronal tissue has a lot of autofluorescence.
I would suggest you extracted the maximum amount of lipids/lipofuscins etc. possible, by washing sections with methanol, acetone, even xylene, as if you were doing a dehydration/rehydration procedure. You can additionally remove free aldehydes washing with Tris/glycine solution, or with NH4Cl solution or with Sodium Borohydride solution 1mg/ml.
This should reduce backgroung considerably.
Good luck!
I am pretty sure you have seen this link, but just in case you have not found it. . .
http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf
It talks about reducing the lipofuscin background.
The other possibility is from PFA itself. In that case, you can use a reducing agent, such as NaBH4 or simply shorten the fixation time. Using a freshly prepared PFA also helps.
Good luck.
Fixative induced fluorescence caused by aldehyde fixatives is predominantly in the green/yellow spectral range. In basic terms it's due to the change in strucutre caused by the cross linking and reaction of the fixative. Things with lots of chemical bonds such as C=C also produce a lot of autofluorescence.
There is a good guide here to different causes and cures: http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf
My advice would be to try a different aldehyde free fixative like methanol or reduced aldehyde such as Zamboni's or PLP. People generally find these result in less autofluorescence.
An alternative startegy is to pre-photobelach your samples before staining. This works but has mixed effects on antigens.
Thanks all, comments much appreciated. As a first step, we're going to make a comparison of young vs. old animals and 4% PFA vs. 2% PFA/0.2% picric acid and see what turns out best...
We had a lot of problems with autofluorescence in human pineal and pituitary gland samples. What appeared to help was Sudan black staining. The method is described here: http://onlinelibrary.wiley.com/doi/10.1111/j.1600-079X.2011.00880.x/abstract;jsessionid=AD81553549178960E0878F7462A44BDD.f03t02 and here: http://press.endocrine.org/doi/abs/10.1210/en.2012-2274?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed.
Good luck, Erik
Thanks Erik.
We tried some young vs. old mice and it seems that lipofuscin accumulation is to blame.
Thanks again everyone!
Hello Ewan:
In my own personal experiences with tissue samples, where autofluorescence is a big problem, I used Sod. Borohydride at 5mg/ml conc for 5, 10 or 15 min to see which was more effective in reducing the background and it helped. I strongly believe the time for tissue fixation with 4%paraformaldehyde is not enough. You could go up to an hour and then wash them off extensively with PBS buffer before cryo-sectioning. Hope it helps. Good luck.
My 2 cents in addition to the advises above: You mention that young is better than old and lipofuschin might be to blame. Usually, the oxidized lipofuschin is the worse offender. Make sure that the surface of the slice is never exposed to air. Bubbling all solution with N2 (under a draft for PFA !!) before use may also very much reduce the background. This can be combined with other treatments. I hope this helps.
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