Have you tried doing the two antibodies in reverse order? Sometimes the two antigens are configured such that the binding of antibodies to one blocks the access of antibodies to the other. Picture it as one antigen protruding farther from the cell surface than the other. If you stain the "taller" antigen first, it can form a protective shield, so to speak, that prevents antibodies from penetrating to the underlying "shorter" antigen. But, if you stain the "shorter" antigen first, then the "taller" one may still protrude far enough for its anitibody to find it.
Histonet mailing list