In situ hybridization using non-isotopically labelled synthetic oligonucleotide probes : application in clinical pathology

Many years after first being reported, the practice of in situ hybridization remained confined to the research laboratory. The reasons for this restriction were mainly due to the hazardous nature of the isotopic nucleotides used to label nucleic acid probes in combination with their poor cellular resolution and cost. The coincidental advent of several different research reagents led to an increase in the application of in situ hybridization in the mid 1980's. Introduction of non-isotopic nucleic labels combined with easily accessible probes, obtained either by cloning techniques or automated synthesis, and developments in immunocytochemistry, enabled in situ hybridization to progress to routine use for the first time. The methods involving the use of non-isotropic probes have been as numerous as the reports themselves, with little attempt being made to establish a routine methodology. A standard, adaptable technique must be deduced so as to place routine in situ hybridization alongside immunocytochemistry in contemporary histopathology. In this study individual in situ hybridization parameters such as probe labelling, tissue handling, target unmasking, stringency washing, and hybrid detection have been investigated. This investigation has enabled the formulation of a standard technique which has then been applied in several clinical pathological studies. With minor adaptations the technique has been applied to formalin fixed paraffin embedded tissue, cryostat sections and cytocentrifuge preparations. The finding of these studies are reported in this thesis.