Multiple immunolabeling with antibodies from the same host species in combination with tyramide signal amplification

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method with primary antibodies originating from the same host species. When primary antibodies are raised in the same host species, the secondary species-specific antibodies can cross-react with each of the primary antibodies. This obstacle can however be avoided with the use of striping buffers eluting the primary/secondary antibody complex. After elution of the previous primary/secondary antibody complex, the next primary antibody from the same host species can be applied. Recently, a group from VENTANA (Tucson, AZ, USA) presented a fully automated multiplex protocol for fluorescent immunohistochemistry on the platform of VENTANA's BenchMark ULTRA slide stainer using the same species antibodies in combination with tyramide signal amplification. We adapted the automated protocol of VENTANA for the use in a routine histochemical laboratory and present here a standard procedure with a manual mode of operation for simultaneously detecting two or more antigens from the same host species.