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Author Topic: autofluorescence  (Read 16410 times)

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Offline apple

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autofluorescence
« on: November 24, 2005, 08:49:04 PM »
Hi,

Aldehyde fixatives react with amines and proteins to generate fluorescent products.
Will FFPE sections have fixative-induced fluorescence? Can I find out section is autofluorescent or not after dewaxing in xylene & washing in PBS?

Thanks

Apple

autofluorescence
« on: November 24, 2005, 08:49:04 PM »

Offline ImmunoNYC

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Re: autofluorescence
« Reply #1 on: November 24, 2005, 10:09:37 PM »
Hi Apple, Just like frozen sections you can merely do as you suggest which is prepare your tissue to PBS put on a coverslip and look at it and *voila* you will see the autofluorescence.

It *is* expected that you will get higher autofluorescence from FFPE sections due to just the chemistry that you have mentioned. Even so, not all tissues or fixation preparations will always give you bad background and it is worth doing a case by case basis. I routinely "quench" autofluorescence using glycine for 30 minutes before commencing my staining protocol (but after rehydration) to quench freee aldehydes.
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Offline apple

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quench autofluorescence
« Reply #2 on: November 28, 2005, 05:49:44 PM »
Hi MaximinaNYC,

Could you please give me the detailed protocl for quenching aldehyde induced autofluorescence using glycine solution? what concentration? pH? for how long? at room temperature, isn't it?

How could I find out if the section contains endogenous autofluorescence or fixative induced autofluorescence?  If the section contains both type of autofluorescence, how to quench these autofluorescence?

Many thanks

Apple

Offline apple

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autofluorescence
« Reply #3 on: December 13, 2005, 10:13:37 AM »
Hi,

I just read some reference about quenching autofluorescence.
The FFPE sections I use most likely contain Lipofuscin-like autofluorescence.
For Lipofuscin-like autofluorescence, CuSO4 in ammonium acetate buffer and Sudan Black solution are offen used for quenching autofluorescence. Has anybody used these treatment before?
Should I do this treatment before or after IF staining? If I want to do DAPI or PI nuclar counterstaining, should I do the quench treatment just after secondary ab and before applying counterstain ?

Can somebody give me some suggestion?

Many thanks

Apple

Offline apple

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urgent!!!
« Reply #4 on: December 14, 2005, 09:33:35 PM »
Can anybody help about quenching autofluorescence here? Should I use Sudan Black B solution before or after counter staining with DAPI or PI?

It's urgent. Please help.

Many thanks

Offline ImmunoNYC

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Re: urgent!!!
« Reply #5 on: December 15, 2005, 12:25:54 AM »
In a previous post on some related topic I gave you a reference to a paper which does just this. Did you look at the methods section for a protocol?

Offline apple

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autofluorescence
« Reply #6 on: December 15, 2005, 10:16:31 AM »
Hi, MaximinaNYC

Yes. I did read the reference paper you recommended. However all of them don't mention about nuclear counterstain.  

They suggest to use Sudan Black B solution after the secondary antibody, then wash off SB solution and mount the slides. However for my experiment, I will use PE conjugated primary ab and counterstain with DAPI. My DAPI counterstain is from Vector and it is called DAPI mounting solution. So when I apply the DAPI counterstain, actually I already mounted the slide. So I can't move coverslip to apply Sudan Black B again. What's your suggestion???

Thank

Apple

Offline apple

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autofluorescence
« Reply #7 on: December 15, 2005, 10:19:30 AM »
My question is will DAPI be affected by Sudan Black B solution?

Many thanks

Apple

Offline ImmunoNYC

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autofluorescence
« Reply #8 on: December 15, 2005, 10:58:13 AM »
Ok, I didn't understand your question I guess. So I would suggest since DAPI is in your mounting medium you don't have much of a choice but to wash it off and then counterstain. That is what I would do myself anyway. I am not 100% sure though.

I hope that since you are using a directly labeled primary that your signal is very intense. The cards are stacked against you using a direct primary and much less one labeled with PE which is not photostable. No amplification and with a unreliable fluorophore.

Offline apple

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autofluorescence
« Reply #9 on: December 15, 2005, 01:28:20 PM »
Hi MaximinaNYC,

Yes. I also think this primary ab is not a good one for immunofluorescent application. This ab is original for Flow cytometry and FACS application. So could I use this primary ab with horse anti mouse secondary ab and HRP label system for IHC assay instead?  Will the PE conjugation affect binding of secondary antibody?

Thanks

Apple

Offline ImmunoNYC

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autofluorescence
« Reply #10 on: December 18, 2005, 12:12:30 AM »
Yes. You could also use a rhodamine, Alexa 555, CY3 or similarly labeled secondary to detect, although it wouldn't be as amplified as using DAB etc.
Quote from: "apple"
So could I use this primary ab with horse anti mouse secondary ab and HRP label system for IHC assay instead?  Will the PE conjugation affect binding of secondary antibody?

Offline apple

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autofluorescence
« Reply #11 on: December 19, 2005, 05:39:50 AM »
Hi, MaximinaNYC

For example, I have Biotin labelled horse anti mouse secondary antibody and Avidin labelled FITC. So I could also use these agents with the PE conjugated mouse primary antibody. Am I right?

Thanks

Apple

Offline ImmunoNYC

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autofluorescence
« Reply #12 on: December 19, 2005, 05:26:22 PM »
Yes, I don't see why not. Just make sure to include proper controls.

autofluorescence
« Reply #12 on: December 19, 2005, 05:26:22 PM »