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,