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  • 1
    Electronic Resource
    Electronic Resource
    Simultaneous triple fluorescence detection of mRNA localization, nuclear DNA, and apoptosis in cultured cells using confocal scanning laser microscopy (1997)
    Springer
    Histochemistry and cell biology 108 (1997), S. 307-311 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  We describe a multifluorescence labeling technique for simultaneous detection of mRNA, nuclear DNA, and apoptosis in cultured cells. Digoxigenin-labeled cRNA probes were used to study proto-oncogene expression in rat pleural mesothelial cells undergoing apoptosis following exposure to crocidolite asbestos or hydrogen peroxide (H202). Hybridized cRNA probe was detected by immunolocalization with an anti-digoxigenin monoclonal primary and fluorophore-conjugated anti-mouse secondary antibody. Cells undergoing apoptosis were simultaneously identified by the TdT-mediated biotin-dUTP nick-end labeling (TUNEL) method and a streptavidin-conjugated far-red fluorophore, and nuclear DNA was stained with oxazole yellow dimer (YOYO-1). With confocal scanning laser microscopy, we demonstrated increased c-jun mRNA expression within the cytoplasm of both TUNEL-positive and non-apoptotic cells following exposure to either crocidolite asbestos or H202. Thus, this technique represents a useful in vivo approach for evaluating apoptosis-associated gene expression with confocal scanning laser microscopy.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004180050170
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  • 2
    Electronic Resource
    Electronic Resource
    Contrasting of Lowicryl K4M thin sections (1990)
    Springer
    Histochemistry and cell biology 95 (1990), S. 123-136 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00266584
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  • 3
    Electronic Resource
    Electronic Resource
    Three-dimensional reconstruction by confocal laser scanning microscopy in routine pathologic specimens of benign and malignant lesions of the human breast (1997)
    Springer
    Histochemistry and cell biology 107 (1997), S. 267-278 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Confocal laser scanning microscopy (CLSM) has become an exciting new instrument because of its increased resolution over conventional wide-field microscopy and its high performance three-dimensional (3D) optical sectioning. Although CLSM has been used extensively in cell biology, few applications have been reported in routine clinical pathology. In this study, 3D reconstruction was performed on routine formalin-fixed, paraffin-embedded tissues of normal mammary duct, simple ductal hyperplasia, intraductal papillary hyperplasia, ductal carcinoma in situ, invasive carcinoma, and lymph node metastatic carcinomas of the human breast by using computer-assisted CLSM in conjunction with a 3D reconstruction software package (microVoxel). The selected specimens were sectioned at 30 μm, mounted on glass slides, and stained with the DNA fluorescent probe, YOYO-1 iodide. The nuclear DNA and chromatin texture were clearly demonstrated after pretreatment with RNAase and hydrolysis with 2 N HCl. High quality 3D images were obtained by processing the optical section stacks with volume render and surface display parameters in microVoxel. 3D morphologic characteristics of different breast lesions were examined in various orientations by angular image rotation. The clearly benign lesions (simple ductal hyperplasia and intraductal papillary hyperplasia) revealed similar 3D morphologic features, including: (1) smooth nuclear surface and homogeneous chromatin fluorescence intensity; (2) hyperplastic cell nuclei showing similar shape and volume; and (3) clear-cut margin of basement membrane defined by spindle-shaped myocytes of the ductal outer layer. In contrast, carcinomas displayed remarkably different features in 3D morphology, including: (1) irregular nuclear surface; (2) marked nuclear pleomorphism (irregular, angulated and indented shape of nuclear volume); (3) irregular and coarse chromatin texture; (4) chaotic arrangement of tumor cell nuclei; and (5) absence of myocytes, indicating no clear margin at the site of infiltration of cancer cells. In conclusion, nuclear structure, specifically demonstrated by CLSM of YOYO-1 iodide fluorescently stained cells, used in tandem with 3D volume morphologic reconstruction, may provide a useful research diagnostic tool in pathology.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004180050112
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  • 4
    Electronic Resource
    Electronic Resource
    Improved quantitative characterization of atherosclerotic plaque composition with immunohistochemistry, confocal fluorescence microscopy, and computer-assisted image analysis (2000)
    Springer
    Histochemistry and cell biology 113 (2000), S. 161-173 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  To quantitatively characterize contributions of major constituents to the composition of a given atherosclerotic plaque, we have developed an approach employing immunohistochemistry, confocal scanning laser microscopy, and computer-assisted image analysis. The method developed permits identification of plaques that are particularly vulnerable to rupture and elucidation of the nature of the composition of a given plaque, as well as the extent of luminal encroachment. Thus, it should be useful in experimental animals and ultimately in patients in delineating compositional changes in response to potentially deleterious genetic and environmentally induced factors and to potentially therapeutic interventions designed to diminish plaque vulnerability.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004180050435
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  • 5
    Electronic Resource
    Electronic Resource
    Prevention of non-specific interactions of gold-labeled reagents on tissue sections (1989)
    Springer
    Histochemistry and cell biology 92 (1989), S. 47-56 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The protein A-gold technique is amongst the most useful labeling techniques available for light and electron microscopic immunolabeling. Some electron microscopic studies, however, have suggested that protein A-gold, and other protein-gold complexes as well, may bind non-specifically to certain tissue structures, particularly in skin, creating a specious pattern of labeling. We utilized the protein A-gold technique with antiserum to both involucrin and keratin under a variety of conditions to document the specificity of labeling. When the standard conditions were followed, the protein A-gold technique produces highly specific results. These conditions include: 1. the blocking of unreacted aldehyde groups by amination; 2. the blocking of non-specific binding sites on tissue sections by preincubation with inert proteins; and 3. the use of proper concentration of the protein A-gold complex. However, non-specific labeling could be produced if the three components of the standard protocol were omitted. In particular, the use of too concentrated protein A-gold lead to non-specific labeling. We report here also updated working protocols for antigen detection with protein A-gold on semithin Lowicryl K4M and paraffin sections which provide optimal staining results.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00495015
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  • 6
    Electronic Resource
    Electronic Resource
    Light microscopical detection of antigens and lectin binding sites with gold-labelled reagents on semi-thin Lowicryl K4M sections: Usefulness of the photochemical silver reaction for signal amplification (1987)
    Springer
    Journal of molecular histology 19 (1987), S. 235-245 
    Details
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary In the present study, we have investigated the applicability of semi-thin sections from low temperature Lowicryl K4M-embedded tissues for cytochemical labelling with protein A—gold and lectin—gold complexes. In order to ensure the best possible signal-to-noise ratio antibodies, protein A—gold and lectin—gold were applied in concentrations used for labelling at the electron microscope level. Furthermore, due to the lack of an appropriate chemical procedure for resin removal, untreated semi-thin sections were incubated. Under such conditions, semi-thin sections displayed either no visible staining or only a faint incomplete staining. However, following photochemical silver reaction, the latent or faint incomplete staining was rendered visible in most cases. It is concluded that the same block of Lowicryl K4M-embedded tissue and the same labelling reagents can be used for both light and electron microscopical cytochemical studies. At the light microscopical level, a high degree of structural and specific staining information is obtained. The reactivity of cellular components with antibodies or lectins is preserved even after years of storage of the blocks or slides containing semi-thin sections.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01680634
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  • 7
    Electronic Resource
    Electronic Resource
    Elderberry bark lectin-gold techniques for the detection of Neu5Ac (α2,6) Gal/GalNAc sequences: applications and limitations (1988)
    Springer
    Journal of molecular histology 20 (1988), S. 478-490 
    Details
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The lectin from the elderberry (Sambucus nigra L.) bark, shown to recognize the sequence neuraminic acid (α2,6) galactose/N-acetylgalactosamine, was applied for detecting binding sites in Lowicryl K4M sections by light and electron microscopy. The lectin was used either directly complexed to colloidal gold or in a two-step cytochemical affinity technique. The lectin-gold complex proved to be superior and thus was extensively tested on rat liver, kidney and hepatoma cells as well as on sheep and bovine submandibular glands. Controls to establish specificity of lectin-gold binding included sugar and glycoprotein inhibition tests and enzymic removal of sialic acid. In agreement with biochemical data demonstrating the potentiating effect of sialic acid on the binding of the lectin to oligosaccharides, enzymic removal of sialic acid from liver sections resulted in abolition of lectin staining. However, in the submandibular glands, neuraminidase pretreatment of the sections had no effect on the subsequent lectin-gold binding. In rat kidney some structures became negative while others retained the lectin-gold staining due to binding to penultimate.N-acetylgalactosamine exposed after sialic acid removal. In line with this, spot blot analysis demonstrated that the lectin-gold complex reacted with both fetuin and asialofetuin. Taken together, these results suggest that, for cytochemical staining, the sialic acid and the galactose/N-acetylgalactosamine lectin combining subsites ofSambucus nigra L. lectin are equally reactive with cellular glycoconjugates and that neuraminidase predigestion of tissue sections is of utmost importance to ensure specificity of staining for the sequence neuraminic acid (α2,6) galactose/N-acetylgalactosamine.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01002646
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  • 8
    Electronic Resource
    Electronic Resource
    Intracellular localization of parvovirus B19 nucleic acid at the ultrastructural level by in situ hybridization with digoxigenin-labelled probes (1993)
    Springer
    Journal of molecular histology 25 (1993), S. 421-429 
    Details
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Conditions suitable for immunogold detection of digoxigenin-labelled DNA probes hybridized to parvovirus B19-infected erythroid cells embedded in Lowicryl K4M and LR White acrylic resins were established at the electron microscope level. The protocol was initially optimized using a positive control probe for whole human DNA which produced signal over the heterochromatin of all nucleated cells. In cultures harvested 2 days postinfection, B19 nucleic acid was detected mainly within the centrinuclear region of erythroid cells exhibiting characteristic margination of the chromatin. The B19 hybridization signal was largely unaffected by denaturation and was resistant to RNase digestion but sensitive to DNase digestion, indicating that it was mainly single-stranded B19 DNA. Relatively few gold particles were found over crystalline arrays of viral capsids, consistent with the observation that they are composed of mainly ‘empty’ capsids. B19 nucleic acid was detected in apparent transit from nucleus to cytoplasm through pores in the nuclear membrane. While the sensitivity of this system is limited by the fact that hybridization occurs only at the surface of the section, it is a rapid and specific means of localizing viral nucleic acids with a high degree of resolution.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00157806
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