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Concentration of fluorescent secondary antibody


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#1 Rumpel

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Posted 11 February 2011 - 03:57 AM

Hello there,

I want to do immunohistochemistry with fluorescent secondary antibodies. My problem is, that I have no idea in which concentrations I should use the fluorescent secondary antibodies because for conventional immunohistochemistry we use the Thermo UltraVision Quanto Detection System and in this system the secondary antibody is just called 'link' and I have no idea of what concentration this link is.

Are there some typical concentrations for secondary antibodies that I should try out?

Thank you very much in advance,
Rumpel

#2 Rsm

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Posted 11 February 2011 - 05:54 AM

1:500 dilution. Don't know in mg/ml, but always works for me... But that is for confocal imaging.
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#3 steffi333

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Posted 01 March 2011 - 02:14 AM

Hello there,

I want to do immunohistochemistry with fluorescent secondary antibodies. My problem is, that I have no idea in which concentrations I should use the fluorescent secondary antibodies because for conventional immunohistochemistry we use the Thermo UltraVision Quanto Detection System and in this system the secondary antibody is just called 'link' and I have no idea of what concentration this link is.

Are there some typical concentrations for secondary antibodies that I should try out?

Thank you very much in advance,
Rumpel



First see if the antibody you're using comes with any references or a manufacturer's recommendation. This should give you a good range to work with.

Then the best thing to do would be to design an experiment to observe at which concentration you get the least background signal.

Carry out a series of stains using different concentrations of your secondary while keeping the concentration of your primary constant. E.g. try 1 in 50, 1 in 100, 1 in 200, 1 in 500, 1 in 1000 etc.

Also carry out the same stains WITHOUT the primary antibody but WITH the secondary (i.e. a no 1st Ab control).

Your ideal secondary concentration will one at which you get:
a.) a good signal WITH the primary
...and...
b.) no (or very weak) signal WITHOUT the primary.




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