An immunoenzyme triple-staining method using both polyclonal and monoclonal antibodies from the same species. Application of combined direct, indirect, and avidin-biotin complex (ABC) technique.

For immunohistological analysis, simultaneous detection of multiple cellular epitopes, as compared to single staining of serial sections, is sometimes needed. Therefore, immunoenzyme triple-staining protocols were tested with polyclonal and monoclonal antibodies on tissue sections and cytospin preparations. Various immunoconjugates were used in different combinations of methods, of which not all proved to be suitable. Of the tested protocols, one yielded superior results for both monoclonal and polyclonal antibodies, with optimal preservation of their original avidity. The method consists of a combination of indirect, direct, and avidin-biotin complex technique. The three antigens can be distinguished clearly and selectively by the reaction products of the enzyme activities of beta-galactosidase (green), alkaline phosphatase (blue), and horseradish peroxidase (red).