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 sen...

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Bibliographic Details
Published in:The journal of histochemistry and cytochemistry 1994-08, Vol.42 (8), p.1071-1077
Main Authors: Kerstens, HM, Poddighe, PJ, Hanselaar, AG
Format: Article
Language:English
Subjects:
Biological and medical sciences
Blood Cells - chemistry
Diverse techniques
DNA Probes
Fundamental and applied biological sciences. Psychology
Humans
Immunohistochemistry
In Situ Hybridization - methods
Interphase
Metaphase
Microscopy - methods
Molecular and cellular biology
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Summary: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.
ISSN:0022-1554
1551-5044
DOI:10.1177/42.8.8027526