Multiple immunoenzyme labeling using heat treatment combined with the polymer method: An analysis of the appropriate inactivation conditions of primary antibodies

Multiple immunoenzyme labeling is of considerable value to detect several antigens in the same specimen, although this technique is limited when the primary antibodies have been raised in the same animal species. Multiple immunoenzyme labeling using heat treatment is a simple, reliable and straightforward technique wherein the heat treatment prevents mixed labeling and cross-reaction. The present study determined the inactivation time for primary antibodies by heat treatment in order to apply this procedure to routine histopathological diagnosis and research, and found that the inactivation time differed among the primary antibodies. The secondary antibodies and the labeling enzyme were completely inactivated by heating for 10 min. Therefore, the inactivation of the primary antibodies is crucial to perform multiple immunoenzyme labeling using heat treatment. The sequential combination of the primary antibodies is also important; in the study presented here, an anti-thyroid transcription factor-1 (TTF-1) antibody should be used first and anti-cytokeratin AE1/AE3 antibody second, but not in the opposite sequence, to avoid a mixed-colour-labeling reaction. The present data provided the optimum combination of primary antibodies for multiple immunoenzyme labeling using heat treatment.