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) technique, using biotin- and digoxigenin-labeled chromosome-specific DNA probes. First, several immunochemical detection systems were optimized and compared for sensitivity and simultaneous applicability. Two non-interfering immunochemical systems were chosen for simultaneous detection of the DNA probe labels. This resulted in combination of an alkaline phosphatase (AP)-conjugated avidin-biotin system with a horseradish peroxidase (HRP)-conjugated antibody system for detection of biotin- and digoxigenin-labeled DNA probes, respectively. Development of AP with New Fuchsin-naphthol phosphate and HRP with diaminobenzidine-H2O2 resulted in stable, well-contrasting (red and black, respectively) color precipitates visible by conventional light microscopy. The double-target ISH technique was successfully applied on a wide variety of biological materials, such as metaphase spreads, cytospin, and Thin-prep samples of cytological specimens, frozen tissue sections, and formalin-fixed, paraffin-embedded tissue sections. In particular, on tissue sections, 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 chromosome imbalances.
Department of Pathology, University Hospital Nijmegen, The Netherlands.
H M
HM
Kerstens
P J
PJ
Poddighe
A G
AG
Hanselaar
D001773:Blood Cells / Q000737:chemistry; D015342:DNA Probes; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D007399:Interphase; D008677:Metaphase; D008853:Microscopy / Q000379:methods
D015342:DNA Probes