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In situ hybridisation of EBV DNA-DNA hybrids using wet heat in polypropylene containers
1Department of Virology, Royal Postgraduate Medical School, Hammersmith Hospital, London.
Aims:
To explore procedures designed to optimise DNA-DNA in situ hybridisation, using cells infected with Epstein-Barr virus (EBV) and tissues and subfragments of the EBV DNA as probes.
Methods:
The denaturation step occurred in a polypropylene container, using wet heat generated by a hot water container, the pressure cooker, or the microwave oven, without coverslips, reaching a temperature of 121 degrees C or more in these two last systems. Two different visualisation systems were used.
Results:
Fixed cells and tumours harbouring a high and medium to low copy number (a few hundreds to 33 copies per cell), were clearly labelled, using a simple reiterated subfragment (BamW) of the EBV DNA, and fresh frozen cells, harbouring a very low copy number (one to two on average) labelled using BamW as well as BamH (single non-reiterated 6 kilobase subfragment).
Conclusion:
This is a valuable alternative technique for DNA-DNA ISH that can be used in fresh frozen samples as well as fixed samples.