Concentration dependent and adverse effects in immunohistochemistry using the tyramine amplification technique

Although the tyramine amplification technique to enhance sensitivity in immunohistochemistry has been described in numerous methodological papers, it has not yet gained access to diagnostic immunohistochemistry. This is mainly due to problems and pitfalls occurring in adaptation of this method to routine application. In this study a monoclonal antibody and a polyclonal antiserum (pan-cytokeratin and anti-myoglobin) were tested in tissues with different amounts of epitopes, using a checkerboard table and testing a total of 133 different dilution combinations of both the tyramide solution and the primary antibodies. The specific tissue investigated, i.e. the amount of accessable epitope to be detected and the applied concentration of the tyramide solution mainly influenced the staining reaction. Several pitfalls such as an uneven distribution of the staining or dramatic overstaining (paradoxical overstaining) must be considered to achieve optimal results. In conclusion, our data confirm methodological studies that the tyramine amplification technique is a powerful method to enhance immunohistochemical sensitivity. However, for reliable daily practice several pitfalls of the technique have to be circumvented.