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autofluorescence

autofluorescence - Protocols and Methods Forum

autofluorescence - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 11-11-2009, 09:44 AM
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Default autofluorescence



hello everybody
I'm doing a PhD in horse virology and it is more than 4 months that I can not develop an efficient protocol for Immunofluorescence of infected PBMC cells
I am using a monoclonal antibody that binds to a nucleocapsid protein. This product is available on the market
my problem is that this mAb binds aspecifically neutrophils (you can identify them for the morphology of the nucleus ... the really strange thing is that using an irrelevant isotype autofluorescence does not develop!
I tried to use a protocol to limit autofluorescence but was not successful
I do not know what to do, time passes and I always stop at the same point

I hope you can help

thanks
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Old 11-11-2009, 02:29 PM
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Default Re: autofluorescence

Are you purifying the PBMC's? If not you may want to do so in order to avoid contamination. Neutrophils have a large number of Fc receptors, and those may be binding your antibody. Alternatively, the antibody may be recognising an unrelated epitope on the neutrophils.

There are a couple of options:
1) Stain your neutrophils so you can ID them and then eliminate them later from your analysis. There are various neutrophil-specific antibodies you can use for this task (anti-GR1 for example).

2) Purify your prep to remove the contaminating cells. This is rather easy - plate down your prep on a tissue culture dish for 8hrs or so, then wash. Neutrophils should not stick, and will wash off.

3) Buy, or make, your antibody as a Fab or F(ab)2. This will eliminate Fc receptor binding

4) Pretreat your cells with an isotype antibody from the same species (and isotype) as your primary antibody. This should block any Fc interactions, but may activate your cells.

5) Block your cells with serum from the same (or closely related, if same is not available) species as your primary antibody. This should reduce any non-specific binding.

6) Try another antibody, if one is available. Ideally another clone, but even the same clone from a different company can behave differently.

Bryan
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