DOUBLE-TARGET IN-SITU HYBRIDIZATION IN BRIGHTFIELD MICROSCOPY

For brightfield detection of two different DNA target sequences in one sample, we developed a double-target in situ hybridization (ISH) tech nique, using biotin- and digoxigenin-labeled chromosome-specific DNA p robes. First, several immunochemical detection systems were optimized and compared for sensitivity and simultaneous applicability. Two non-i nterfering immunochemical systems were chosen for simultaneous detecti on of the DNA probe labels. This resulted in combination of an alkalin e phosphatase (AP)-conjugated avidin-biotin system with a horseradish peroxidase (HRP)-conjugated antibody system for detection of biotin- a nd digoxigenin-labeled DNA probes, respectively. Development of AP wit h New Fuchsin-naphthol phosphate and HRP with diaminobenzidine-H2O2 re sulted in stable, well-contrasting (red and black, respectively) color precipitates visible by conventional light microscopy. The double-tar get ISH technique was successfully applied on a wide variety of biolog ical materials, such as metaphase spreads, cytospin, and Thin-prep sam ples of cytological specimens, frozen tissue sections, and formalin-fi xed, paraffin-embedded tissue sections. In particular, on tissue secti ons, where quantitative interpretation of ISH data can be hampered by truncation of nuclei, the double-target ISH technique appeared to be a valuable tool for demonstration of chromosome aberrations and chromos ome imbalances.