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How to quench glutaraldehyde fluorescence?
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+ Fluorescence
+ Glutaraldehyde
+ Fluorescence quenching
+ Immunocytochemistry
+ Chemistry
+ Immunofluorescence
+ Fixed cell
+ Autofluorescence
Posted by
Kazakh Wazaky
How to quench glutaraldehyde fluorescence?
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
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
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)
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)
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
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
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.
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.
Hello, I would like to test new antibodies by immunofluorescence with glutaraldehyde 4% fixation and I also have the problem of high autofluorescence. Having no NaBH4, I was wondering if your use of H2O2 Przemysław Zakrzewski was successful? and does glycine really not work? Thanks
Hello, I would like to test new antibodies by immunofluorescence with glutaraldehyde 4% fixation and I also have the problem of high autofluorescence. Having no NaBH4, I was wondering if your use of H2O2 Przemysław Zakrzewski was successful? and does glycine really not work? Thanks
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
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
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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)
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)
More
VOTE
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
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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VOTE
Hi,
You can also try 50mM Glycine in 1X PBS. It works for my immunocytochemistry experiments.
Regards,
Ekta
Hi,
You can also try 50mM Glycine in 1X PBS. It works for my immunocytochemistry experiments.
Regards,
Ekta
More
VOTE
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.
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.
More
VOTE
Hello, I would like to test new antibodies by immunofluorescence with glutaraldehyde 4% fixation and I also have the problem of high autofluorescence. Having no NaBH4, I was wondering if your use of H2O2 Przemysław Zakrzewski was successful? and does glycine really not work? Thanks
Hello, I would like to test new antibodies by immunofluorescence with glutaraldehyde 4% fixation and I also have the problem of high autofluorescence. Having no NaBH4, I was wondering if your use of H2O2 Przemysław Zakrzewski was successful? and does glycine really not work? Thanks
More
VOTE