Intensification of 3,3'-diaminobenzidine precipitation using the ferric ferricyanide reaction, and its application in the double-immunoperoxidase technique.

As in double-immunoperoxidase methods, colour mixing usually indicates unwanted interactions between reagents of the first and second sequences, it is desirable to prevent such superimposition of colours by eliciting adequate colour intensity in the first immunoperoxidase sequence. The brown oxidation product of 3,3'-diaminobenzidine (DAB) in the first immunoperoxidase sequence can be intensified by applying the ferric ferricyanide reaction, resulting in intense greenish-blue staining. When the primary antibody is used at a sufficient concentration, cells labelled in the first sequence do not cross-react with the red chromogen, 3-amino-9-ethylcarbazole (AEC), used in the second sequence. Thus, this double-immunoperoxidase method results in different cell populations being clearly labelled in contrasting colours. Primary antibodies from the same species and the same type of link antibodies can be used in the two separate immunoperoxidase sequences. When primary antibodies raised in different species and two types of link antibodies are used, the method can, without loss of sensitivity, be shortened by performing the first two incubation steps simultaneously.