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  • 1
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The extent of chromosomal mosaicism in human preimplantation embryos was examined using an improved procedure for the preparation and spreading of interphase nuclei for use in fluorescence in situ hybridisation, allowing the analysis of every nucleus within an embryo. One cell showed no hybridisation signals in only three of the 38 embryos that were included in this study, i.e. the hybridisation efficiency per successfully spread nucleus was 99% (197/200). Double-target in situ hybridisation analyses with X- and Y-chromosome-specific probes was performed to analyse nine embryos resulting from normal fertilisation, 22 polypronucleate embryos and seven cleavage-stage embryos where no (apronucleate) or only one pronucleus (monopronucleate) was observed. We also analysed autosomes 1 and 7 by double-target in situ hybridisation in the nuclei of two apronucleate, one monopronucleate and four polypronucleate embryos. All nine embryos that resulted from normal fertilisation were uniformly XY or XX. None of the apronucleate or monopronucleate embryos was haploid: three were diploid, one was triploid and three were mosaic. Fertilisation was detected by the presence of a Y-specific signal in four of these embryos. Of the polypronucleate embryos, two were diploid, two were triploid and 18 were mosaic for the sex chromosomes and/or autosomes 1 and 7. These results demonstrate that fertilisation sometimes occurs in monopronucleate embryos and that chromosomal mosaicism can be detected with high efficiency in apronucleate, monopronucleate and polypronucleate human embryos using fluorescence in situ hybridisation.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 677 (1993), S. 0 
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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  • 3
    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.
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  • 4
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  A selection of normal human tissues was investigated for the presence of lamins B1, B2, and A-type lamins, using a panel of antibodies specific for the individual lamin subtypes. By use of immunoprecipitation and two-dimensional immunoblotting techniques we demonstrated that these antibodies do not cross-react with other lamin subtypes and that a range of different phosphorylation isoforms is recognized by each antibody. The lamin B2 antibodies appeared to decorate the nuclear lamina in all tissues examined, except hepatocytes, in which very little lamin B2 expression was observed. In contrast to previous studies, which suggested the ubiquitous expression of lamin B1 in mammalian tissues, we show that lamin B1 is not as universally distributed throughout normal human tissues as was to be expected from previous studies. Muscle and connective tissues are negative, while in epithelial cells lamin B1 seemed to be preferentially detected in proliferating cells. These results correspond well with those obtained for lamin B1 in chicken tissues. The expression of A-type lamins is most prominent in well-differentiated epithelial cells. Relatively undifferentiated and proliferating cells in epithelia showed a clearly reduced expression of A-type lamins. Furthermore, most cells of neuroendocrine origin as well as most hematopoietic cells were negative for A-type lamin antibodies.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Histochemistry and cell biology 99 (1993), S. 141-149 
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Expression patterns of intermediate filament proteins have been studied during early mouse embryo development. For this purpose, pre-implantation embryos at different stages of development after in vitro fertilization were studied using antibodies to cytokeratins, vimentin and lamins, using the indirect immunofluorescence assay. The levels of expression were quantitated and localization of the protein constituents was assessed by means of confocal scanning laser microscopy. Our studies showed that, although the embryos grew in culture, vimentin could not be detected in a filamentous organization. Immunofluorescence for cytokeratins was only positive from the 8-cell stage onwards. In the morula stage an increased level of cytokeratin expression was observed with a transitional staining pattern, combining a filamentous and a diffuse occurrence. In the blastocyst stages profound cytokeratin filaments were seen in trophoblast cells but not in the inner cell mass. When the cytokeratin subtypes were analysed separately, it became apparent that expression levels of cytokeratins 8 and 18 increased gradually up to a filamentous pattern in the blastocyst stage. Cytokeratins 7 and 19, although elevated in the latter stage and showing a filamentous distribution, were not found as prominently as cytokeratins 8 and 18. A-type as well as B-type lamins could be detected in all developmental stages examined, as a faintly reactive nuclear lamina. In blastocysts both lamin types were detected in trophoblast as well as in inner cell mass.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Molecular biology reports 19 (1994), S. 1-1 
    ISSN: 1573-4978
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Molecular biology reports 19 (1994), S. 31-44 
    ISSN: 1573-4978
    Keywords: interphase cytogenetics ; chromosome aberrations ; cancer ; DNA probes ; comparative genomic hybridization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1573-2657
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Established myogenic cell lines of different species and tissue origin have been used to study expression and organisation of muscle-specific proteins during differentiation. Furthermore, primary cultures of rat myocard cells were used to examine these same processes during dedifferentiation. In particular, we were interested in the general mechanism that underlies the changes in the supramolecular organisation of titin during in vitro myogenesis. It became obvious that in the differentiating muscle cell cultures the redistribution of desmin, actin and myosin in a typical, differentiation state dependent fashion, always showed a certain delay when compared to titin. The sequence of changes in the assembly of cytoskeletal and sarcomeric structures observed during differentiation of the cell lines was reversed during the process of dedifferentiation in cultured rat myocard cells. These results all indicate that titin is an early marker of myogenic differentiation, both in vivo and in vitro, and that the typical reorganisation of this giant molecule is independent of species or muscle cell type.
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  • 9
    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.
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  • 10
    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.
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