NONISOTOPIC 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 hydribization kit and describe the favorable results gained with the amended protocol, which ape as follows: 1. The application of a de creased amount of both the probe and the chromogen substrate did not a lter the quality of reactions. Therefore we were able to make 60 react ions 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 ord er of hybrid detection and posthybridization washing, we succeeded in removing the excess amount of probe-ABC-AP-BAAV-ABC-AP conglomerates w ithout losing the target sequence. 4. Using alkaline phosphatase or AB C-AP-BAAV-ABC-AP complex instead of peroxidase it was possible to demo nstrate a very low number of gene copies, even if they were not detect able following the original instructions.