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  • 1
    Electronic Resource
    Electronic Resource
    Direct visualization of bipolar myosin filaments in stress fibers of cultured fibroblasts (1989)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 12 (1989), S. 150-156 
    Details
    ISSN: 0886-1544
    Keywords: stress fibers ; fibroblasts ; myosin ; bipolar filaments ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The authors examined the molecular organization of myosin in stress fibers (microfilament bundles) of cultured mouse embryo fibroblasts. To visualize the organization of myosin filaments in these cells, fibroblast cytoskeletons were treated with gelsolin-like protein from bovine brain (hereafter called brain gelsolin), which selectively disrupts actin filaments. As shown earlier [Verkhovsky et al., 1987], this treatment did not remove myosin from the stress fibers. The actin-free cytoskeletons then were lightly sonicated to loosen the packing of the remaining stress fiber components and fixed with glutaraldehyde.Electron microscopy of platinum replicas of these preparations revealed dumbbell-shaped structures of approximately 0.28 μm in length, which were identified as bipolar myosin filaments by using antibodies to fragments of myosin molecule (subfragment I and light meromyosin) and colloidal gold label. Bipolar filaments of myosin in actin-free cytoskeletons were often organized in chains and lattices formed by end-to-end contacts of individual filaments at their head-containing regions. Therefore, after extraction of actin, it was possible for the first time to display bipolar myosin filaments in the stress fibers of cultured cells.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970120304
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    The use of colloidal metal particles in protein blotting (1987)
    Weinheim : Wiley-Blackwell
    Electrophoresis 8 (1987), S. 403-409 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two procedures are described for staining the overall protein pattern on transfer membranes. Both methods involve the use of colloidal metal particles. AuroDye staining is based on the interaction of anionic colloidal gold particles with cationic proteins at low pH. The method is highly sensitive, but limited to nitrocellulose and polyvinylidene difluoride membranes. FerriDye comprises an incubation in a cationic iron (hydrous) oxide sol and subsequent intensification with potassium hexacyanoferrate in acidic medium. FerriDye is of intermediate sensitivity, but there are no limitations concerning membranes. It can be used on nitrocellulose-, nylon-and polyvinylidene difluoride-based membranes. An immunodetection procedure is described which uses colloidal gold-labelled secondary antibodies to visualize antigen-antibody interactions. This signal can be significantly enhanced by silver. The procedure explained uses a new light-insensitive and user-friendly silver enhancement system.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150080907
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    Konfokale Fluoreszenzmikroskopie in der Zellbiologie (1991)
    Weinheim : Wiley-Blackwell
    Biologie in unserer Zeit 21 (1991), S. 19-25 
    Details
    ISSN: 0045-205X
    Keywords: Life and Medical Sciences
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Der Zellbiologe ist daran interessiert, spezifische Strukturen in Zellen sichtbar zu machen, urn mit dem Mikroskop die Morphologie zu untersuchen. Dabei macht er sich die Eigenschaften von Antikörpern zunutze, die bestimmte Moleküle innerhalb der Zelle erkennen und gezielt an ihnen binden. Um den Bindungsort der spezifischen Antikörper sichtbar zu machen, wird ein zweiter Antikörper zugefügt, der an die Oberfläche des ersten Antikörpers bindet. Dieser zweite Antikörper ist mit einem fluoreszierenden Molekül gekoppelt, das mit Licht einer kürzeren Wellenlänge angeregt wird und Licht einer längeren Wellenlänge wieder ausstrahlt (Abbildung 1). Mit Filtern lassen sich Anregung und Abstrahlung trennen, und die markierten Strukturen der Zelle leuchten in einem Fluoreszenzmikroskop hell auf. Die hier beschriebene Technik wird als indirekte Immunfluoreszenz bezeichnet, da erst der zweite Antikörper fluorochromiert ist.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/biuz.19910210109
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  • 4
    Electronic Resource
    Electronic Resource
    Ultrathin cryosections in the plane of cell monolayers: Evaluation of their potential for antibody localization studies of the cytoskeleton (1988)
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 8 (1988), S. 391-399 
    Details
    ISSN: 0741-0581
    Keywords: Ultracryomicrotomy ; Immunogold ; Cultured cells ; Negative staining ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In spite of the fact that the preembedding method is satisfactory for the ultrastructural localization of cytoskeletal proteins, there is a need for a localization method that retains the cells' ground substance, delicate filament arrangements, and membrane-filament interactions and provides a good delineation of ultrastructural detail. Ultracryomicrotomy, a resinless sectioning method, can combine good morphology with optimal antibody labeling. Until now, however, it has not been possible to section cell monolayers parallel to their plane of growth. This is a prerequisite for the localization of proteins along segments of filaments, contained within the section thickness. We describe such a method and give a first appreciation of its potential for antibody localization studies of cytoskeletal proteins. The method consists of seeding cells on a parallel 0.75-mm-thick gelatin substrate that can later be cut and used as a mounting block. An adapted negative staining has yielded a very useful delineation of the well-preserved structures within the cells, even in combination with immunogold labeling. The latter has been in its indirect version less satisfactory in dense microfilament bundles because of penetration problems, and more satisfactory on microtubules. Clearly, the penetration properties of gold probes will have to be improved before this method will become widely applicable. The availability of a sectioning method like this will provide the basis for further progress. There will be many cases which will justify the use of this relatively more difficult approach.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jemt.1060080409
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    Paper (German National Licenses)
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  • 5
    Electronic Resource
    Electronic Resource
    The three-dimensional architecture of the mitotic spindle, analyzed by confocal fluorescence and electron microscopy (1991)
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 18 (1991), S. 61-73 
    Details
    ISSN: 0741-0581
    Keywords: Fluorescence microscopy techniques ; Poleward chromosome movement ; Microtubule dynamics ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Fluorescence microscopy techniques have become important tools in mitosis research. The well-known disadvantages of fluorescence microscopy, rapid bleaching, phototoxicity and out-of-focus contributions blurring the in-focus image are obstacles which still need to be overcome. Confocal fluorescence microscopy has the potential to improve our capabilities of analyzing cells, because of its excellent depth-discrimination and image processing power. We have been using a confocal fluorescence microscope for the study of the mechanism of poleward chromosome movement, and report here (1) a cell preparation technique, which allows labeling of fixation sensitive spindle antigens with acceptable microtubule preservation; (2) the use of image processing methods to represent the spatial distribution of various labeled elements in pseudocolour; (3) a novel immunoelectron microscopic labeling method for microtubules, which allows the visualization of their distribution in semithin sections at low magnification; and (4) a first attempt to study microtubule dynamics with a confocal fluorescence microscope in living cells, microinjected with rhodamine labeled tubulin.Our experience indicates that confocal fluorescence microscopy provides real advantages for the study of spatial colocalization of antigens in the mitotic spindle. It does not, however, overcome the basic limits of resolution of the light microscope. Therefore, it has been necessary to use an electron microscopic method. Our preliminary results with living cells show that it is possible to visualize the entire microtubule network in stereo, but that the sensitivity of the instrument is still too low to perform dynamic time studies. It will be worthwhile to further develop this new type of optical instrumentation and explore its usefulness on both fixed and living cells.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jemt.1060180110
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