The Experts below are selected from a list of 216 Experts worldwide ranked by ideXlab platform
Nicholas J Severs - One of the best experts on this subject based on the ideXlab platform.
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Freeze-fracture Cytochemistry: a simplified guide and update on developments.
Journal of Microscopy, 1991Co-Authors: Nicholas J SeversAbstract:SUMMARY A wide variety of methods by which Cytochemistry and freeze-fracture can be successfully combined have recently become available. All these techniques are designed to provide information on the chemical nature of structural components revealed by freeze-fracture, but differ in how this is achieved, in precisely what type of information is obtained, and in which types of specimen can be studied. Colloidal gold labelling is the most widely used cytochemical technique in freeze-fracture Cytochemistry, and for many of the methods it is indispensable. In principle, there are four points in which the cytochemical labelling step may be integrated into the standard freeze-fracture procedure: (i) before the specimen has been frozen, (ii) after it has been fractured and thawed, (iii) after platinum shadowing or (iv) after completion of the full replication sequence. Retention of the gold label so that it can be viewed with replicas can be achieved by depositing platinum and/or carbon upon the labelled surface, thereby partially entrapping the marker particles within the replica, or by retaining, attached to the replica, fragments of fractured membrane (or other cellular components) that would normally have been lost during the replica cleaning step. Another approach to visualizing the label is to use sections, either with portions of a replica included face-on, or for examining the fracture path through the sample (without replica). Recent developments have centred on the use of replicas to stabilize half-membrane leaflets; not only may these and associated attached components be retained for labelling just before mounting, but they provide a means for manipulating the specimen— specifically, turning it over during processing—so that additional structural information can be obtained. This article aims to explain how modern freeze-fracture cytochemisty works, and how the various techniques differ in what they can tell us about membranes and other cellular structures. With the effectiveness of many of the techniques now demonstrated, freeze-fracture Cytochemistry is firmly established, alongside a range of related labelling techniques, for increasing application in cell and membrane biology in the 1990s.
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Freeze-fracture Cytochemistry: a simplified guide and update on developments.
Journal of microscopy, 1991Co-Authors: Nicholas J SeversAbstract:A wide variety of methods by which Cytochemistry and freeze-fracture can be successfully combined have recently become available. All these techniques are designed to provide information on the chemical nature of structural components revealed by freeze-fracture, but differ in how this is achieved, in precisely what type of information is obtained, and in which types of specimen can be studied. Colloidal gold labelling is the most widely used cytochemical technique in freeze-fracture Cytochemistry, and for many of the methods it is indispensable. In principle, there are four points in which the cytochemical labelling step may be integrated into the standard freeze-fracture procedure: (i) before the specimen has been frozen, (ii) after it has been fractured and thawed, (iii) after platinum shadowing or (iv) after completion of the full replication sequence. Retention of the gold label so that it can be viewed with replicas can be achieved by depositing platinum and/or carbon upon the labelled surface, thereby partially entrapping the marker particles within the replica, or by retaining, attached to the replica, fragments of fractured membrane (or other cellular components) that would normally have been lost during the replica cleaning step. Another approach to visualizing the label is to use sections, either with portions of a replica included face-on, or for examining the fracture path through the sample (without replica). Recent developments have centered on the use of replicas to stabilize half-membrane leaflets; not only may these and associated attached components be retained for labelling just before mounting, but they provide a means for manipulating the specimen--specifically, turning it over during processing--so that additional structural information can be obtained. This article aims to explain how modern freeze-fracture Cytochemistry works, and how the various techniques differ in what they can tell us about membranes and other cellular structures. With the effectiveness of many of the techniques now demonstrated, freeze-fracture Cytochemistry is firmly established, alongside a range of related labelling techniques, for increasing application in cell and membrane biology in the 1990s.
Toshihiro Takizawa - One of the best experts on this subject based on the ideXlab platform.
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Freeze-fracture Cytochemistry: a new fracture-labeling method for topological analysis of biomembrane molecules.
Histology and histopathology, 2000Co-Authors: Toshihiro Takizawa, John M. RobinsonAbstract:Freeze-fracture Cytochemistry allows visualization of cellular and molecular characteristics of biomembranes in situ. In this review, we discuss freezefracture Cytochemistry with special reference to a new cytochemical labeling of replicas, the detergentdigestion fracture-labeling technique. In this procedure, unfixed cells are rapidly-frozen, freeze-fractured, and physically stabilized by evaporated platinum/carbon. The frozen cells are then removed from the freezefracture apparatus to thaw and are subsequently treated with detergents. After detergent-digestion, replicas are labeled with cytochemical markers. We demonstrate that the technique is a versatile tool for direct analysis of the macromolecular architecture of biomembranes and allows identification of particular intracellular membrane organelles. In addition, we demonstrate the application of ultrasmall gold to freeze-fracture immunoCytochemistry. Freeze-fracture Cytochemistry is a valuable technique for investigating topology and dynamics of membrane molecules.
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5 -Nucleotidase in Rat Photoreceptor Cells and Pigment Epithelial Cells Processed by Rapid-freezing Enzyme Cytochemistry
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 1998Co-Authors: Toshihiro TakizawaAbstract:This report describes the subcellular distribution of 5'-nucleotidase (5'-NT) in rat photoreceptor cells and pigment epithelial cells processed by rapid-freeze enzyme Cytochemistry. There was a striking difference in the ultrastructural localization of 5'-NT activity between rod outer segments after freeze-substitution fixation and conventional fixation. By rapid-freezing enzyme Cytochemistry, 5'-NT activity was localized in the extradiscal space of intact nonvacuolated discs, whereas by conventional Cytochemistry it was shown in the intradiscal space of artifactual vacuolated discs. In the freeze-substituted retinal cells, an appreciable difference in functional 5'-NT molecules was also found. The soluble 5'-NT on the cytoplasmic side of the disc membrane was vital in the rod outer segments, whereas the membrane-bound ecto-5'-NT on the exoplasmic (external) surface of the apical process was active in the pigment epithelial cells. Rapid-freezing enzyme Cytochemistry should be worth employing as a method to reveal the fine localization of enzyme activity at the level of cell ultrastructures, which are poorly preserved by conventional fixation, and should provide information approximate to that in living cells.
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Freeze-fracture Cytochemistry: a new method combining immunoCytochemistry and enzyme Cytochemistry on replicas.
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 1998Co-Authors: Toshihiro Takizawa, Takuma Saito, John M. RobinsonAbstract:We describe a new freeze-fracture cytochemical technique consisting of combined immunoCytochemistry and enzyme Cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphates activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.
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Freeze-Fracture Enzyme Cytochemistry Reveals the Distribution of Enzymes in Biological Membranes : Enzyme Cytochemical Label-Fracture and Fracture-Label
ACTA HISTOCHEMICA ET CYTOCHEMICA, 1997Co-Authors: Toshihiro Takizawa, Eiko Nakazawa, Takuma SaitoAbstract:During the past decade, freeze-fracture Cytochemistry, the combination of freezefracture electron microscopy with Cytochemistry, has greatly contributed to the investigation of the macromolecular architecture of biological membranes. The application of enzyme cytochemical labeling technique to freeze-fracture Cytochemistry (i. e., freeze-fracture enzyme Cytochemistry) has not received considerable attention although by enzyme cytochemical labeling technique many enzyme molecules can be labeled with metal cations and enzyme Cytochemistry has been of great utility in cell biology studies. In this study, we report freeze-fracture enzyme Cytochemistry: “label-fracture method” and “fracture-label method”. Freeze-fracture enzyme Cytochemistry was applied to the study of acid phosphatase, 5′-nucleotidase in the proximal tubular epithelium of the rat kidney, ectoadenosine triphosphatase in human neutrophils and alkaline phosphatase in rat neutrophils, and was shown to be a technique that visualized these enzyme activities on replicas. Cerium as the capture agent was a useful enzyme cytochemical probe in this technique. Lead was applicable for labeling of plasma membrane-associated enzyme molecules. This technique leads to new applications that may extend the usefulness of freeze-fracture Cytochemistry for the analysis of biomembrane structure.
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Freeze-Fracture Enzyme Cytochemistry: Application of Enzyme Cytochemistry to Freeze-Fracture Cytochemistry
Journal of electron microscopy, 1996Co-Authors: Toshihiro Takizawa, Takuma SaitoAbstract:We present a novel freeze-fracture Cytochemistry method based upon enzyme Cytochemistry. By this method, freeze-fractured membranes are labeled with cerium as an enzyme cytochemical marker on replicas. The cerium capture method was suitable for freeze-fracture enzyme Cytochemistry because the cerium phosphate reaction product is stable after replica-cleaning. As a model system, acid phosphatase, which is a well-known lysosomal marker, was detected on freeze-fractured membranes of lysosomes in the proximal tubular epithelium of the rat kidney. This technique should be a useful addition for analyzing the ultrastructure of biological membranes.
John M. Robinson - One of the best experts on this subject based on the ideXlab platform.
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Freeze-fracture Cytochemistry: a new fracture-labeling method for topological analysis of biomembrane molecules.
Histology and histopathology, 2000Co-Authors: Toshihiro Takizawa, John M. RobinsonAbstract:Freeze-fracture Cytochemistry allows visualization of cellular and molecular characteristics of biomembranes in situ. In this review, we discuss freezefracture Cytochemistry with special reference to a new cytochemical labeling of replicas, the detergentdigestion fracture-labeling technique. In this procedure, unfixed cells are rapidly-frozen, freeze-fractured, and physically stabilized by evaporated platinum/carbon. The frozen cells are then removed from the freezefracture apparatus to thaw and are subsequently treated with detergents. After detergent-digestion, replicas are labeled with cytochemical markers. We demonstrate that the technique is a versatile tool for direct analysis of the macromolecular architecture of biomembranes and allows identification of particular intracellular membrane organelles. In addition, we demonstrate the application of ultrasmall gold to freeze-fracture immunoCytochemistry. Freeze-fracture Cytochemistry is a valuable technique for investigating topology and dynamics of membrane molecules.
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Freeze-fracture Cytochemistry: a new method combining immunoCytochemistry and enzyme Cytochemistry on replicas.
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 1998Co-Authors: Toshihiro Takizawa, Takuma Saito, John M. RobinsonAbstract:We describe a new freeze-fracture cytochemical technique consisting of combined immunoCytochemistry and enzyme Cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphates activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.
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Rapid-freezing Cytochemistry: preservation of tubular lysosomes and enzyme activity.
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 1991Co-Authors: John M. Robinson, M J KarnovskyAbstract:We show that tubular structures present in phorbol ester-stimulated macrophages are sensitive to commonly used chemical fixatives (i.e., they usually become fragmented during fixation). These structures are well preserved in macrophages that are physically fixed by rapid-freezing and subsequent freeze-substitution in osmium-acetone. We have developed methods that combine rapid-freezing, freeze-substitution, and enzyme Cytochemistry for preservation of these tubular structures and for detection of endocytosed material (i.e., horseradish peroxidase). This method of rapid-freeze Cytochemistry may be useful in other situations where chemical fixation does not adequately preserve cell structures, particularly of membrane compartments.
Daniel Catovsky - One of the best experts on this subject based on the ideXlab platform.
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The classification of acute leukaemia.
Leukemia, 1992Co-Authors: Daniel Catovsky, Estella MatutesAbstract:The standard methods for classifying acute leukaemias now include morphology, Cytochemistry and membrane markers. Major advances in immulogy, in particular the development of monoclonal antibodies (MCAb) with lineage specificity, have provided objective positive criteria for the diagnosis of acute lymphoblastic leukaemia (ALL). The FAB group has recognised the importance of MCAb for the classification of some forms of acute myeloid leukaemia (AML), such as megakaryoblastic leukaemia, AML-M7, in which reactivity with MCAb against platelet glycoproteins is a requirement for diagnosis. More recently the group has defined a type of myeloblastic leukaemia with minimal differentiation, AML-M0, in which myeloid Cytochemistry is negative and the diagnosis is made by the expression of myeloid antigens and negative lymphoid markers in the blast cells
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The role of an anti-myeloperoxidase antibody in the diagnosis and classification of acute leukaemia: a comparison with light and electron microscopy Cytochemistry.
British journal of haematology, 1992Co-Authors: Valeria Buccheri, Estella Matutes, V. Shetty, N. Yoshida, Ricardo Morilla, Daniel CatovskyAbstract:The enzyme myeloperoxidase (MPO) is the hallmark of the myeloid lineage. We have analysed the presence of MPO in blasts from 180 cases of acute leukaemia (103 acute myeloid leukaemia (AML) and 77 acute lymphoid leukaemia (ALL) by means of monoclonal antibodies anti-MPO and immunoCytochemistry (alkaline phosphatase anti-alkaline phosphatase method). The aim of the study was to investigate the specificity and sensitivity of this marker compared with MPO Cytochemistry by light (LM) and electron microscopy (EM), and with the expression of myeloid antigens. Anti-MPO was positive (greater than 3% blasts) in all but one of the 90 AML positive by LM Cytochemistry. Of 13 AML cases negative by MPO Cytochemistry, six showed 3-10% blasts reactive with anti-MPO and were also positive with antibodies to CD13 and/or CD33. The presence of MPO was confirmed in four of these by EM. The overall positivity of anti-MPO in AML was 92%. Anti-MPO was negative in all but two ALL (6% and 8% positive blasts). The blasts in these two cases were also CD13, CD33 and MPO positive by EM; both were thus reclassified as biphenotypic. Another two ALL reinterpreted as biphenotypic were negative by MPO Cytochemistry and anti-MPO but were MPO positive by EM and with CD13 and/or CD33. We conclude that anti-MPO is a sensitive and specific early marker of myeloid blasts and should be incorporated in the routine immunophenotyping of acute leukaemia.
Gustavo D. Aguirre - One of the best experts on this subject based on the ideXlab platform.
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Diethylene glycol distearate (DGD): a versatile embedding medium for retinal Cytochemistry
Journal of neuroscience methods, 1993Co-Authors: Jun C. Huang, Kristina Mieziewska, Nancy J. Philp, Theo Van Veen, Gustavo D. AguirreAbstract:Embedment in diethylene glycol distearate (DGD) was shown to be highly desirable and versatile for retinal cytochemical studies, including in situ hybridization, immuno- and lectin Cytochemistry. This method allows for preservation of fine tissue detail as well as good reaction sensitivity. It appears to be more suitable than most other methods currently used for light microscopic retinal Cytochemistry.
