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Author Topic: need help urgently - autofluorescence  (Read 7464 times)

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Offline apple

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need help urgently - autofluorescence
« on: December 13, 2005, 07:32:34 PM »
Hi everybody,

I have some FFPE sections. After de-waxing in xylene, I observed them under fluorescent microscope and found they all have autofluorescence. (I think they are Lipofuscin like autofluorescence because my sections are brain sections). I will use 0.001%-0.1% Sudan Black B to quench autofluorescence. I directly stain these sections with PE lablled mouse monoclone antibody. Then counterstain with DAPI.
I am worrying about following problems:

1. Because PE is a very miserable fluorescent label, I am afraid that Sudan Black solution will bleach fluorescent labelled signal. So after incuation with PE labelled ab, I want to observe slide under fluorescent microscope first. If I can see very strong autofluorescent signal I will use Sudan Black B solution to quench them. If I won't see strong autofluorescent signal, I can continue counterstain with DAPI. Is this OK?

2. Because I do not have enough samples to do more trials (different Sudan Black B concentrations, different primary ab concentrations, high temperature antigen retrieval method etc.). What's the best experiment design? (I mean to use less sample and get more data/results).

Many thanks

Apple

need help urgently - autofluorescence
« on: December 13, 2005, 07:32:34 PM »

Offline apple

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need help urgently - autofluorescence
« Reply #1 on: December 14, 2005, 09:37:06 PM »
Please!!!! Has somebody done quench autofluorescence before?  Could you please share me some of your experience?

Many thanks

Offline richard03

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need help urgently - autofluorescence
« Reply #2 on: December 14, 2005, 10:16:43 PM »
My suggestion is to use enzyme (HRP or AP) labeled antibody.

Offline apple

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need help urgently - autofluorescence
« Reply #3 on: December 15, 2005, 06:44:08 AM »
Hi, richard03

I have PE conjugated mouse monoclone antibody. I also have Biotin labelled horse anti mouse secondary antibody and Avidin labelled HRP. Shall I try this method? Do you think the PE conjugation will affect secondary ab bind to primary ab?

Many thanks

Apple

Offline richard03

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need help urgently - autofluorescence
« Reply #4 on: December 15, 2005, 08:02:33 PM »
Yes. You can use ABC method for PE conjugated primary antibody.

Richard

Offline ImmunoNYC

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Re: need help urgently - autofluorescence
« Reply #5 on: December 18, 2005, 12:19:01 AM »
FYI, there is a reagent from Chemicon to get rid of autofluorescence called "Autofluorescence Eliminator Reagent". See here:

http://www.chemicon.com/Product/ProductDataSheet.asp?ProductItem=2160


Quote from: "apple"
After de-waxing in xylene, I observed them under fluorescent microscope and found they all have autofluorescence. (I think they are Lipofuscin like autofluorescence because my sections are brain sections).

Offline apple

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need help urgently - autofluorescence
« Reply #6 on: December 19, 2005, 05:28:55 AM »
Hi, MaximinaNYC

Thanks very much.

Apple

Offline ImmunoNYC

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need help urgently - autofluorescence
« Reply #7 on: December 19, 2005, 05:27:13 PM »
I just read there is also one from Molecular Probes. Might be worth checking out. I have not used it myself though so cannot personally vouch for it.

Offline apple

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need help urgently - autofluorescence
« Reply #8 on: December 20, 2005, 06:32:23 PM »
Thanks MaximinaNYC.

need help urgently - autofluorescence
« Reply #8 on: December 20, 2005, 06:32:23 PM »