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  • 1
    ISSN: 0741-0581
    Keywords: Immunocytochemistry ; Protein A-Gold ; Lowicryl ; Glycolmethacrylate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The postembedding protein A-gold immunocytochemical approach has been introduced as an alternative to other techniques for the ultrastructural localization of antigenic sites. The present review deals with the development, the theoretical background, and technical approach of the protein A-gold method as well as the different modifications introduced in order to enhance the resolution of the results and to perform double labelings on the same section. Various examples demonstrate the reliability and the wide range of application of this technique. In addition, some problems, pitfalls, and limitations particular to this method are reported.
    Additional Material: 34 Ill.
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  • 2
    ISSN: 0741-0581
    Keywords: Vascular cell cultures ; Transmission electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A method is described for obtaining optimal, reproducible ultrastructure of vascular smooth muscle cells and vascular endothelial cells in culture. Routinely grown cultures are prepared for TEM with a precise regimen of fixation, postfixation, en bloc staining, dehydration, and embedment. The most important aspects of this procedure are the following: (1) fixation with a percentage-gradient series of glutaraldehyde solutions at 37°C, (2) immediate postfixation with osmium tetroxide solution, and (3) block-staining with uranyl acetate solution to eliminate any extraction of constituents during subsequent processing.
    Additional Material: 8 Ill.
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  • 3
    ISSN: 0741-0581
    Keywords: Monolayer cells ; preparation for SEM ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Monolayers of PtK-1 and HeLa cells grown on glass or plastic supports are extremely susceptible to lacerations, e.g., splits and cracks caused mainly by shrinkage when prepared for scanning electron microscopy (SEM). We find that a four-step fixation procedure including glutaraldehyde, OsO4, tannic acid, and uranylacetate application, in combination with critical point drying, drastically reduces these structural damages. In addition, the conductivity of the specimens is enhanced, so that they can be investigated without gold coating. Transmission electron microscopy (TEM) investigation of perpendicular sections in the area of lacerations provides evidence that the subcortical cytoskeletal elements are of crucial importance in maintaining cell membrane stability during the preparations. Our relatively quick and simple procedure results in an improved structural appearance of the cells.
    Additional Material: 3 Ill.
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  • 4
    ISSN: 0741-0581
    Keywords: Opioids ; Receptors ; Brain ; Radioautography ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Two met-enkephalin analogs (FK 33-824 and FW 34-569, Sandoz) were utilized for in vitro labeling of opioid binding sites in the rat central nervous system. Binding kinetics determined in 20-μm-thick frozen tissue sections of the striatum revealed that both pentapeptides bind to a single population of sites at 20°C with an apparent dissociation constant (KD) of approximately 1-2 nM and a maximum capacity (B max) of 65-170 fmoles/mg protein. Radioautographic data suggest that this population is the same for iodinated and tritiated forms of the FK compound and the iodinated FW analog. Fixation of labeled sections with high concentrations of glutaraldehyde allowed proportional retention of more than 50% of specifically bound 125I-FK molecules in all brain regions after histological processing for high-resolution radioautography. In contrast, glutaraldehyde fixation did not prevent the loss of bound 125I-FW molecules. These differences are attributed to the presence in FK, but not in FW molecules, of a free primary amino group considered essential for cross-link formation between aldehydes and proteins, and imply that a majority of FK-receptor complexes may be stabilized by glutaraldehyde. Consistent with this observation is the fact that the radioautographic distribution of specifically bound 125I-FK was unchanged after fixation and dehydration. In electron microscopic radioautographs prepared from prefixed, vibratome-cut striatal sections that were incubated with 125I-FK and fixed with glutaraldehyde, silver grains were found to be mostly associated with neuronal plasma membrane interfaces. The present methodological approach thus appears to be compatible with electron microscopic localization of opioid binding sites in the central nervous system and might be applicable to the localization of other types of binding sites using radioligand molecules that contain a free primary amino group.
    Additional Material: 9 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0741-0581
    Keywords: Ceramics ; Electron microscopy ; Ion milling ; Specimen preparation ; Sputtering ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Ion bombardment to perforation is a common technique in the materials sciences by which thin specimens can be prepared for transmission electron microscopy. The process is not without complication and involves radiation damage to the specimen and tends not to preserve the initial specimen topology. Some of the more important facets of the ion-milling process, pertinent to such specimen preparations, are described.
    Additional Material: 9 Ill.
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  • 6
    ISSN: 0741-0581
    Keywords: Low-temperature SEM ; Direct SEM-viewing ; Living plant specimens ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Young fresh Tradescantia reflexa stamen hair cells were used to clarify the optimal conditions for direct viewing and taking photographs with a scanning electron microscope (SEM) equipped with a cryo-system. The rate of protoplasmic streaming in the cells was measured under an optical microscope after examining and photographing them in the SEM over a period of a few minutes. Almost the same rate of streaming (5.5 μm/second, 20°C) was observed in nonirradiated control cells and irradiated cells photographed in the SEM using an accelerating voltage of 10 kV with the cryo-stage at a temperature of - 15°C. (The specimen holder and specimen were not at this temperature, but, rather, probably somewhat higher.) Fresh plant organs, tissues, and cells were also tested under the same conditions. The fine structure was well preserved in detail. The procedures were as follows: (1) prompt attachment of fresh plant materials on an aluminum specimen holder with double-faced adhesive Scotch tape or a small amount of plastic adhesive for woodcraft; (2) setting the holder on the cryo-stage cooled to -15°C in advance and rapid evacuation; and (3) quick SEM examination and photography (within several minutes). The advantages of this method are summarized as follows: (1) high possibility of viewing living materials; (2) minimal artifacts: freedom from chemical fixation and additional procedures utilized in ordinary SEM specimen preparation; and (3) simplicity, speediness, and economy in preparation for viewing. Since the specimens were not likely to be frozen during quick examination and photography, this method might well be called “low-temperature SEM” (LT-SEM) as distinguished from “cryo-SEM”.
    Additional Material: 19 Ill.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0741-0581
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Additional Material: 1 Ill.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0741-0581
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Additional Material: 2 Ill.
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  • 9
    ISSN: 0741-0581
    Keywords: Folds ; Wrinkles ; Epoxy resin ; Thin section ; Botanical specimen ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Fold or wrinkles appear frequently in thin sections cut from botanical material embedded in epoxy resin such as Epon 812 (Polysciences) or EM-bed-812 (Electron Microscopy Sciences). Small wrinkles may occur more or less perpendicular to the cutting direction and mostly across the cell wall, causing problems in the examination of the ultrastructure. We studied the occurrence of wrinkles and folds by methodological sectioning, with regard to block hardness, use of Formvar-support film, and chloroform vapor. Methodological sectioning or changing the block hardness did not reduce the occurrence of wrinkles in sections. Chloroform vapor improved the result as did also the use of uncoated grid. Wrinkles and folds could be avoided completely by embedding the specimens in LX-112 (Ladd) or ERL-4206 (Polysciences) low-viscosity epoxy resins, in which case Formvar-coated grids could be used, and chloroform vapor was not necessary.
    Additional Material: 6 Ill.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0741-0581
    Keywords: Fore-vacuum evaporation ; Carbon films ; Thickness-to-voltage dependence ; Electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Fore-vacuum (pressure ≥ 3 Pa) evaporation of carbon fibers and rods to thin films has been tested and the resulting thicknesses recorded. A modified sputter coater was used as a vacuum evaporator. The quality of the carbon films was evaluated by bright-field and dark-field electron microscopy. Although high-vacuum-evaporated carbon films are superior in quality, low-vacuum-evaporated carbon films were found fully acceptable for routine work in bright field, for both TEM and SEM purposes. Apart from being time-saving, the method presented has the obvious advantage, in SEM preparations, that carbon coating and metal sputtering can be carried out in the same unit without breaking the vacuum.
    Additional Material: 6 Ill.
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