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multistaining with labeled and unlabeled ABs

multistaining with labeled and unlabeled ABs - Flow Cytometry Forum

multistaining with labeled and unlabeled ABs - Flow Cytometry Forum. Discuss FACS cell sorting, flow cytometry, analysis and cell staining.


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Old 01-07-2010, 03:14 PM
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Default multistaining with labeled and unlabeled ABs



Hi there,

I would like to do a multistain experiment (three different ABs) on cultured cells. Two of the ABs are unstained, so I will need a secondary AB, the third one is PE-labelled. Is this possible?! Two unstained AB probably get me in trouble, no? I would probably have to add unstained AB first, wash, then secondary AB, wash, and PE-labelled AB at the end?!

Thanks for your help

Björn
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Old 03-10-2010, 09:24 PM
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Default Re: multistaining with labeled and unlabeled ABs

Staining with multiple primary antibodies and multiple secondaries is certainly possible. However, you must make sure that all secondaries will only bind to their intended primaries. But, don’t forget that the secondaries can stick to each other too, so you must make sure that they are all raised in different animals. If all of your primaries are raise in different animals, and all of your secondaries are compatible, then you can stain using a standard protocol in which you can incubate first in a mixture of primaries followed by a mixture of secondaries.

But what if two or more of your primaries are raised in the same animal? Well, staining for them is still possible. But I must warn you, things will get difficult and messy. You should not expect aesthetic results.

The answer is to do multiple rounds of staining using special secondary antibodies. First, choose a primary antibody that is raised in the same animal as another primary. Incubate first with this antibody, and only this one. Then, incubate with a compatible secondary THAT IS MONOVALENT (Fab only). This is very important, the secondary must be monovalent. If you use a normal bivalent F(ab)2 secondary, then unbound variable regions will be left after your first round of staining. These will then bind to subsequent primary antibodies and you will get false signal. After you stain for one primary/secondary combo, wash the cells like you’ve never washed before. Then incubate with the second primary antibody that was raise in the same animal. Then incubate with the same kind of monovailent secondary antibody, but conjugated to a different color. Once again, wash like there’s no tomorrow. You can then do a third round of staining with additional antibodies (conjugated or not).

Be prepared to perform lengthy optimizations if you go with this method. If you can help it at all, it’s much less trouble to seek out primaries raised in different animals. It’s even preferable to try “weird” ones from that no-name company you may find. But, many times there’s no other option.
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