Concentration of fluorescent secondary antibody - (Feb/11/2011 )
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
-Rumpel-
1:500 dilution. Don't know in mg/ml, but always works for me... But that is for confocal imaging.
-Rsm-
Rumpel on Fri Feb 11 11:57:34 2011 said:
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.
-steffi333-