double immunofluorescence - (Jun/13/2014 )

  we want to see the phosphorylation of kinase in in interneurons. by using kinase antibody and the marker of interneurons.

 the signal of interneurons marker is a bit low. while when we tried  kinase antibody alone, which is coupled to fluorophore, the signal was good.. 

when we do double immunofluroscence with both antibodies, and looking at confocal imaging, we start first looking at cells labeled with interneuron marker, the signal is  low. when we pass to kinase marker, we see signal only in the cells that were also labeled with interneuron marker. 

so , we dont believe much in the result.  we wonder whether the first immuno inhibit the penetration of the second antibody , thereby, non specific signal. 

please if you have suggestions, let me know and thanks in advance,

Hmm - I would guess that the antibodies you are using for this are both the same species (e.g. the kinase and interneuron antibodies are both produced in rabbits) and you are using the secondaries that will pick them both up?

Another scenario is that you are using one of the antibodies in the organism it was raised (e.g. you are using a mouse antibody on a mouse brain).

A bit more detail on what you have done will be helpful.

thank you very much for your reply !

Indeed, both antibodies are rabbit.

the experiment is to see the phosphorylation of kinase in subtype of neurons .double IF, with anti phospho kinase, and anti-somatostatin.

thank you

In that case - there isn't much you can do other than try finding an antibody raised in a different species for one of the proteins or directly conjugating one of the fluorophores to one of the primary antibodies.