In situ hybridization using biotinylated probes. An evaluation of different detection systems

In situ hybridization with biotin-labelled DNA probes is a powerful tool for the detection of viral sequences in infected tissues. However, sensitivity is low when compared to radiolabelled probes. In order to evaluate the impact on staining results of the detection system applied, we have tested various reagents. In our hands the sequential application of streptavidin and biotinylated alkaline phosphatase or development with a monoclonal anti-biotin antibody and the APAAP method gave consistently the best results. Furthermore, nitro blue tetrazolium/bromochloroindolyphosphate seems to be most suitable as a substrate for the alkaline phosphatase in this case. Use of other reagents, especially of a streptavidin-biotinylated alkaline phosphatase complex, resulted in a significantly lower staining intensity.