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  • 1
    Electronic Resource
    Electronic Resource
    Multi-colour brightfield in situ hybridisation on tissue sections (1997)
    Springer
    Histochemistry and cell biology 108 (1997), S. 291-298 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  We describe the brightfield microscopical detection of multiple DNA target sequences in cell and tissue preparations. For this purpose, chromosome-specific DNA probes labelled with biotin, digoxigenin or fluorescein were simultaneously hybridised and detected by enzyme cytochemistry using two horseradish peroxidase (PO) reactions and one alkaline phosphatase (APase) reaction. For triple-colour detection on single cell preparations, the combination of the enzyme precipitates PO/diaminobenzidine (DAB, brown colour), APase/fast red (FR, red colour) and PO/tetramethylbenzidine (TMB, green colour) resulted in an accurate detection of DNA targets. Embedding of the preparations in a thin cross-linked protein layer further stabilised the enzyme reaction products. For in situ hybridisation on tissue sections, however, this detection procedure showed some limitations with respect to both the stability of the APase/FR and PO/TMB precipitates, and the sequence of immunochemical layers in multiple-target procedures. For this reason, the APase/FR reaction was replaced by the APase/new fuchsin (NF, red colour) reaction and the washing steps after the PO/TMB reaction were restricted to the use of phosphate buffer pH 6.0. Furthermore, to improve the efficiency of the ISH reaction, APase/NF was applied in an avidin-biotin complex detection system and, to avoid target shielding in the triple-target ISH, the third primary antibody was applied prior to the second enzyme cytochemical reaction. These adaptations resulted in stable, well contrasting brown, red and green coloured precipitates. After quick haematoxylin counterstaining, the tissue preparations were directly mounted in phosphate buffer and, optionally, embedded in the cross-linked protein layer.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004180050168
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  • 2
    Electronic Resource
    Electronic Resource
    Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors (1999)
    Springer
    Histochemistry and cell biology 112 (1999), S. 89-113 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid sequences (DNA, RNA) in microscopic preparations of tissues, cells, chromosomes, and linear DNA fibers. To date, a wide variety of research and diagnostic applications of ISH have been described, making the technique an integral part of studies concerning gene mapping, gene expression, RNA processing and transport, the three-dimensional organization of the nucleus, tumor genetics, microbial infections, and prenatal diagnosis. In this review, I first describe the ISH procedure in short and then focus on the currently available non-radioactive probe-labeling and cytochemical detection methodologies that are utilized to visualize one or multiple different nucleic acid targets in situ with different colors. Special emphasis is placed on the procedures applying fluorescence and brightfield microscopy, the simultaneous detection of nucleic acids and proteins by combined ISH and immunocytochemistry, and, in addition, on the recent progress that has been made with the introduction of signal amplification procedures to increase the detection sensitivity of ISH. Finally, a comparison of fluorescence, enzyme cytochemical, and colloidal gold silver probe detection systems will be presented, and possible future directions of in situ nucleic acid detection will be discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004180050396
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    Multicolour preparations for in situ hybridization using precipitating enzyme cytochemistry in combination with reflection contrast microscopy (1993)
    Springer
    Histochemistry and cell biology 100 (1993), S. 357-366 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We have further developed a method for the detection of different enzyme cytochemical reaction products by means of reflection contrast microscopy (RCM). By embedding these enzyme precipitates in a protein matrix, we were able to prevent the reaction products from dissolving in immersion oil, which is required for RCM analysis. The applicability of the RCM procedure is, therefore, extended to a range of cytochemical enzyme precipitation methods, which normally result in oil soluble reaction products. To test their usefulness, these enzyme precipitates have been used in single- as well as double-label in situ hybridization (ISH) procedures to visualize a number of DNA target sequences by several different reflection colours, i.e. white, yellow and red. Three repetitive DNA probes for the (sub)centromeric regions of chromosomes 1, 7 and 17, as well as a repetitive DNA probe for the telomeric region of chromosome 1, and two cosmid DNA probes (40 kb each) for both arms of chromosome 11 could be detected with high efficiency in both interphase and metaphase preparations. Moreover the enzyme precipitates were shown to be stable upon exposure to excitation light or upon storage. It may be concluded that these findings render RCM a sensitive method for the visualization of multiple targets in biological specimens.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00268934
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    Paper (German National Licenses)
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  • 4
    Electronic Resource
    Electronic Resource
    Cytochemical detection systems for in situ hybridization, and the combination with immunocytochemistry. ‘Who is still afraid of red, green and blue?’ (1995)
    Springer
    Journal of molecular histology 27 (1995), S. 833-858 
    Details
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary An overview is given of the different non-radioactive cytochemical detection methodologies that are currently utilized to localize nucleic acid sequences in chromosomes, cells and tissue sections. Dependent on the reporter molecule (fluorochrome, enzyme or hapten) that is used to modify the appropriate nucleic acid probe, and the sensitivity that is required, the in situ hybridized sequences can be detected either directly after hybridization or indirectly, using cytochemical detection and amplification layers. These may then contain antibody and/or avidin molecules conjugated to fluorochromes, enzymes or colloidial gold particles. Since the choice of a suitable probe-labelling method in combination with a fluorescence, enzyme cytochemical or immunogold-silver detection procedure is often determined by the user's own practical experience and applications, the different detection methodologies are compared with each other in detail with respect to sensitivity, resolution, applicability for multiple probe detection, and signal evaluation. Furthermore, procedures are reviewed for the combination of in situ hybridization with immunocytochemical detection of proteins and/or incorporated bromodeoxyuridine, which allow the simultaneous visualization of genomic phenotypic and/or cell cycle parameters in the same sample. Possible improvements with respect to sensitivity, specificity and multiplicity of the detection methods, which may be interesting for one's own experimental design, are finally being discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00173839
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    Paper (German National Licenses)
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  • 5
    Electronic Resource
    Electronic Resource
    Cytochemical detection systems forin situ hybridization, and the combination with immunocytochemistry. ‘Who is still afraid of red, green and blue?’ (1995)
    Springer
    Journal of molecular histology 27 (1995), S. 833-858 
    Details
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary An overview is given of the different non-radioactive cytochemical detection methodologies that are currently utilized to localize nucleic acid sequences in chromosomes, cells and tissue sections. Dependent on the reporter molecule (fluorochrome, enzyme or hapten) that is used to modify the appropriate nucleic acid probe, and the sensitivity that is required, thein situ hybridized sequences can be detected either directly after hybridization or indirectly, using cytochemical detection and amplification layers. These may then contain antibody and/or avidin molecules conjugated to fluorochromes, enzymes or colloidial gold particles. Since the choice of a suitable probe-labelling method in combination with a fluorescence, enzyme cytochemical or immunogold-silver detection procedure is often determined by the user’s own practical experience and applications, the different detection methodologies are compared with each other in detail with respect to sensitivity, resolution, applicability for multiple probe detection, and signal evaluation. Furthermore, procedures are reviewed for the combination ofin situ hybridization with immunocytochemical detection of proteins and/or incorporated bromodeoxyuridine, which allow the simultaneous visualization of genomic phenotypic and/or cell cycle parameters in the same sample. Possible improvements with respect to sensitivity, specificity and multiplicity of the detection methods, which may be interesting for one’s own experimental design, are finally being discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02389591
    Library Location Call Number Volume/Issue/Year Availability
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    Paper (German National Licenses)
    Fulltext
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