Patent ID: 8603765

Claim:
A method of detecting presence and/or distribution of three antigens in a sample, comprising: simultaneously contacting a sample with a primary antibody cocktail comprising a buffer aqueous solution of anti-P504S antibody from a first animal species as a first primary antibody, anti-p63 antibody from a second animal species different from the first animal species as a second primary antibody, and anti-high molecular weight cytokeratin (anti-HMWCK) antibody from the second animal species as a third primary antibody, wherein the sample is a tissue or cell, under conditions sufficient for the primary antibodies to specifically bind to antigens in the sample; simultaneously contacting the sample, which has been previously simultaneously contacted with the primary antibody cocktail with a composition comprising a first secondary antibody and a second secondary antibody under conditions sufficient to form secondary antibody complexes with the primary antibodies to antigens in the contacted sample, wherein the first secondary antibody is specific for the anti-P504S primary antibody in the primary antibody cocktail and the second secondary antibody is specific for the rest of the primary antibodies in the primary antibody cocktail, wherein the first secondary antibody is coupled to a poly (alkaline phosphatase) (poly AP) moiety and the second secondary antibody is coupled to a poly (horseradish peroxidase) (poly HRP) moiety, and wherein the composition comprises a buffer for the first and second secondary antibodies; and applying two different chromogens that result in two different colors for separately detecting the bound poly HRP moiety and the bound poly AP moiety on the secondary antibody-contained sample, thereby detecting the secondary antibody complexes on the contacted sample as indicative of the presence and/or distribution of the P504S, p63, and/or HMWCK antigens in the sample, wherein the two different chromogens are (i) 3,3′-diaminobenzidine (DAB) for detecting the poly HRP moiety and (ii) a combination of at least one Fast Red salt with at least one naphthol phosphate (Fast Red) for detecting the poly AP moiety.