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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 15 (1990), S. 81-96 
    ISSN: 0741-0581
    Keywords: Antibody ; GABA ; Glycine ; Acetylcholine ; Auditory system ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Over the last several years our knowledge of neurotransmitter receptors has increased dramatically as receptor types and subtypes have been identified through the development of selective antagonists, neuropharmacological studies, and radioactive ligand binding studies. At the same time major advances were made in the immunocytochemical localization of neurotransmitters and their related enzymes. However, only recently has immunocytochemistry been used to localize neurotransmitter receptors, and these studies have been limited. Four receptors have been localized in the CNS with immunocytochemistry: the nicotinic acetylcholine receptor, the beta-adrenergic receptor, the GABA/benzodiazepine receptor, and the glycine receptor. Of these the glycine receptor has been the most thoroughly characterized. Glycine receptor immunoreactivity is highly concentrated at postsynaptic sites, and the distribution of immunoreactivity appears to correlate closely with glycinergic neurons. However, immunocytochemical studies done on other receptors suggest such a distribution may not always be the case. Some receptors may not be concentrated at postsynaptic sites, and receptor distribution may not always closely fit the distribution of the respective neurotransmitter. Work is rapidly progressing on the purification of other receptors and on the production of selective antibodies which will allow immunocytochemical studies which address these and other questions.
    Additional Material: 7 Ill.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 14 (1990), S. 39-45 
    ISSN: 0741-0581
    Keywords: Exocrine pancreas ; Cryofixation ; Cryomicrotomy ; Freeze-drying ; Freeze-substitution ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: This paper describes and compares the morphology of a relatively complex tissue, the exocrine pancreas, prepared by state-of-the-art anhydrous cryoprocedures. Cryopreparative procedures are being used increasingly for a wide range of applications, for example, electron-probe x-ray microanalysis and immunocytochemical localization of antigenic molecules, because they preserve the composition of the specimen better than procedures involving aqueous media. Some doubts have remained concerning the morphology of cryosections and the precise identification of subcellular structures.We show that thin and sufficiently large cryosections of fresh biological tissues can be produced using commercially available hardware. The freeze-dried cryosections display high intrinsic contrast, are stable under the beam, and allow identification of intracellular fine structure.
    Additional Material: 6 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 14 (1990), S. 348-356 
    ISSN: 0741-0581
    Keywords: Cryofixation ; Cryoprotectant ; Dimethylformamide ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Conditions for cryofixation and freeze-substitution crucial to the ultrastructural preservation of embryonic quail retina were improved. As freeze-substitution makes gentle dehydration and chemical fixation of tissue possible, the suitability of different cryoprotectants were tested in the preceding cryofixation. Additionally, different conditions for chemical prefixation were studied. In cryofixation, all of the “classic” cryoprotectants caused more or less severe tissue destruction. Only dimethylformamide (DMF) and-with certain reservations-dimethylsulfoxide (DMSO) yielded improved structure preservation. Perfusion fixation with a mixture of formaldehyde/glutaraldehyde (FA/GA) was superior to GA alone. In comparison to conventional fixation and dehydration methods, freeze-substitution yielded better ultrastructural preservation of the embryos with fewer artifacts.
    Additional Material: 10 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 15 (1990), S. 2-19 
    ISSN: 0741-0581
    Keywords: Acetylcholine ; Cholinergic fibers ; Electron microscopy ; Ultrastructure ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Cholinergic synapses can be identified in immunocytochemical preparations by the use of monoclonal antibodies and specific antisera to choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine (ACh) and a specific marker for cholinergic neurons. Electron microscopic studies demonstrate that the fibers and varicosities observed in light microscopic preparations of many brain regions are small-diameter unmyelinated axons and vesicle-containing boutons. The labeled boutons generally contain clear vesicles and one or more mitochondrial profiles. Many of these boutons form synaptic contacts, and the synapses are frequently of the symmetric type, displaying thin postsynaptic densities and relatively short contact zones. However, ChAT-labeled synapses with asymmetric junctions are also observed, and their frequency varies among different brain regions. Unlabeled dendritic shafts are the most common postsynaptic elements in virtually all regions examined although other neuronal elements, including dendritic spines and neuronal somata, also receive some cholinergic innervation. ChAT-labeled boutons form synaptic contacts with several different types of unlabeled neurons within the same brain region. Such findings are consistent with a generally diffuse pattern of cholinergic innervation in many parts of the central nervous system. Despite many similarities in the characteristics of ChAT-labeled synapses, there appears to be some heterogeneity in the cholinergic innervation within as well as among brain regions. Differences are observed in the sizes of ChAT-immunoreactive boutons, the types of synaptic contacts, and the predominant postsynaptic elements. Thus, the cholinergic system presents interesting challenges for future studies of the morphological organization and related function of cholinergic synapses.
    Additional Material: 8 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 16 (1990), S. 167-173 
    ISSN: 0741-0581
    Keywords: Plunge-freezing ; Cryofixation ; Freeze-substitution ; Monolayers ; Cultured cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A detailed design for a simple and inexpensive variable-speed (1.0-5.8 m s-1) pneumatic plunge-freezing device is presented. Cultured cells, grown on Formvar-coated 75-mesh gold finder grids, are pneumatically driven into a stirring mixture of propane/isopentane (3:1) cooled by liquid nitrogen (LN2). Premature freezing of the sample in the cryogenic vapors above the cryogen is prevented by plunging through an entry tube into an insulating box, to which a partial vacuum is applied. The cryogenic vapors are drafted into the box at the level of the liquid cryogen by the vacuum, thereby preventing a layer of cold gas from collecting above the cryogen. To prevent the sample from thawing during transfer from the cryogen to the substitution medium, the box top is removed and compressed air is forced through a corrugated tube running the length of the box. The resulting boiling LN2 creates an atmosphere below -120°C in which the transfer can be accomplished.
    Additional Material: 5 Ill.
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