Non-isotopic in situ hybridization of human papilloma virus on histologic sections: an amended protocol

The authors report on their experience with an HPV non-radioactive in situ hybridization kit and describe the favorable results gained with the amended protocol, which are as follows: 1. The application of a decreased amount of both the probe and the chromogen substrate did not alter the quality of reactions. Therefore we were able to make 60 reactions instead of the originally suggested 21. 2. The proteolytic enzyme digestion time could be prolonged by changing proteinase-K for pepsin which intensifies the signal of hybridization. 3. By changing the order of hybrid detection and posthybridization washing, we succeeded in removing the excess amount of probe-ABC-AP-BAAV-ABC-AP conglomerates without losing the target sequence. 4. Using alkaline phosphatase or ABC-AP-BAAV-ABC-AP complex instead of peroxidase it was possible to demonstrate a very low number of gene copies, even if they were not detectable following the original instructions.