Paired indirect immunoenzyme staining with primary antibodies from the same species. Application of horseradish peroxidase and alkaline phosphatase as sequential labels.

Paired indirect immunoenzyme staining based on primary antisera from the same species was performed sequentially without intermediate antibody elution. The first antigen was labelled brown by an immunoperoxidase procedure (either the two-stage indirect method, the unlabelled antibody peroxidase-antiperoxidase method, or the avidin-biotin bridge method using diaminobenzidine (DAB) and hydrogen peroxide as the substrates. The second antigen was labelled blue by applying a two-stage indirect immuno-alkaline phosphatase procedure using naphthol AS phosphate and Fast Blue BB salt as the substrate. In this way, polyclonal mucosal immunocytes were revealed in distinctly contrasting colours when stained for kappa and lambda light chains. Glucagon and somatostatin (D) cells in human pancreatic islets, and gastrin and D cells in human gastric antral glands, were likewise clearly differentiated. Conversely, a mixed colour appeared in some immunocytes after staining for alpha and kappa chains. However, unbalanced colour mixing was sometimes difficult to interpret, and additional experiments demonstrated that unwanted interactions could take place between the two sequences of reagents if the density of the DAB deposits was insufficient. These pitfalls were incompatible with unequivocal double staining in the same cell. Nevertheless, paired staining could be conveniently applied with the described procedures when prior knowledge had established that the antigens in question were located in separate cells.