Double antibody labelling - immunofluorescence - (Mar/25/2015 )

Hi,

When labelling DNA fibres with 2 antibodies (against 2 different nucleotides which would get incorporated at different times) I see labelling of the fibres with both antibodies, which should not happen - why does this occur?

Also, one of the antibodies has much weaker signal than the other, making it difficult to create an overlayed image.

Any suggestions?

Many thanks in advance for your help.

Are the antibodies of the same species? Incubating with antibodies of the same species may lead to binding to the same antigen recognizing the DNA fibers. This is why you see both antibodies labeling the same thing. Also using secondaries that are the same species will lead to unspecific binding as well. Check your conditions and antibodies to make sure they are made in the approproate species.

You may be able to increase your antibody concentration to account for the weaker signal and be able to make your overlay image.