Hello everyone! I would like to ask, how to quench autofluorescence of glutaraldehyde fixed specimen. I always perform standard fixation for immunocytochemistry (4%FA and 0,5%GA) but this time I see, that my specimens embedded in LR Gold is more yellow than usual, which means that GA concentration was higher (I do not have any idea how it is possible, but mistakes happen...). After sectioning I see, that sections have very high autofluorescence in every channel. I found that it is possible to perform immunofluorescence labelling on glutaraldehyde fixed cell after reducing free aldehyde groups using NaBH4, however we do not have this reagent in our lab. I'm thinking whether it is possible to reduce glutaraldehyde using different reducing agent like oxalic acid, or thiosulfate? I'm also thinking about oxidation of aldehyde groups to -COOH, f. ex. with hydrogen peroxide. Actually, I've tried with 3% H2O2, and all fluorescence disappeared, however I don't know if that didn't affect my specimen in some way (I don't want to waste my antibodies before consulting with someone). Glycine and ammonium chloride quenching didn't work.
I would be very greatful for any help provided.
If 3%H2O2 works for you I would go this way. Try several dilutions until you find the minimum that has the effect you are looking for. 3% is fairly high, 0.3% is a concentration used commonly for peroxidase blocking without tissue damage.
NaBH4 treatment is also aggressive with the tissue and when I used it the result wasn't great (poor reduction of autofluorescence)
Dear Przemysław Zakrzewski , may I point you honestly also to the really rich archive files in RG- "Q&A" data base
in using the provided search function (upper menue line to the right, find the clock symbol, left to it a down-pointing arrowhead and SEARCH, left click to the arrowhead, choose "Questions" from the popping menu and insert your search phrase or keywords, like e.g. | autofluorescence quench | which creates the URL: https://www.researchgate.net/search.Search.html?type=question&query=autofluorescence+++quench and - after some second, shows the most (text or word-fitting) results out of Q&A-database.
NB: Searching for | quenching fluorescence glutaraldehyde | creates https://www.researchgate.net/search.Search.html?type=question&query=quenching+fluorescence++glutaraldehyde+ yields similar or partially same, but also other -perhaps interesting results. Best of luck and regards, Wolfgang
Hi,
You can also try 50mM Glycine in 1X PBS. It works for my immunocytochemistry experiments.
Regards,
Ekta
Dear Przemysław Zakrzewski,
glutaraldehyde autofluorescence might be huge and worse than after formaldehyde fixation. How old was your fixative (how did you store the fixative?)?
The best consequence would be to use NON-GA-containing fixatives. Since that might not be possible the only way to find out perhaps is trial and error (I could not find a suited article which describes in particular the successful qenching of GA-AutoFluorescence but some with hints on that, see attachments, but for sure there are many papers out there I do not know yet...). You might find some hints and practical tipps (and hopefully a solution for your problem) in the attached articles out of my literature collection (please - in any case - respect copyright issues by correct citation if you try, apply and finally practically use "knowledge" from those articles.. Thank you) Good luck, best wishes and regards, WM.
If the sample really did contain a lot of Glutaraldehyde as you suspect then it would be very hard to get rid of the fluorescence no matter what you try. Seriously, I fail to understand the scientific basis of fluorescence quenching by H2O2 or Glycine. The reactions of a di-aldehyde such as Glutar can only be reversed with high temp as is done in antigen retrieval. Can you do alternatives such as DAB reaction which would avoid the use of fluorescence? DAB is OK for single channel immunocytochemistry. Remember to use the ABC kit from Vector Laboratories.
For the future try to avoid glutar for immunocytochemistry and fluorescence. With Glutar you may have additional problems of antigen retrieval. The good old neutral buffered formalin is just fine for most immunocytochemistry, I find the use of PFA an overkill most of the time.
Regards
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