Hi Richard,As Gundrun lined out, a sequential method with a heating step in between to remove antibodies from the first staining sequence without affecting the DAB precipitate, is a very safe way to combine two antibodies from the same species. However, with this brown-red color combination one is unable to see sites of co-localization by mixed-color. Recently I published a method in JOH (September 2008) based on two alk phosp methods (Vector Blue and Dako's Liquid Permanent Red) that gives superior results in term of contrast between the basic colors and the purple mixed-color at sites of co-localization. The reaction product of Vector Blue as well as Liquid Permanent Red both survives the heat step without any changes.  Cheers,Chris van der Loos, Amsterdam, Netherlands
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