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Alan T Nurden - One of the best experts on this subject based on the ideXlab platform.
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Ultrastructural analysis of the distribution of von Willebrand factor and fibrinogen in platelet aggregates formed in the PFA-100
Platelets, 1998Co-Authors: Christel Poujol, Alan T Nurden, Paponneau A, Eric Heilmann, Paquita NurdenAbstract:The PFA-100 is a new apparatus used to detect platelet dysfunction in vitro . Anticoagulated blood flows under constant pressure through a capillary, and across an aperture that pierces a membrane coated with collagen and either epinephrine or ADP. Through their ability to adhere and aggregate, platelets occlude the orifice and the closure time is a test of platelet function. Using electron microscopy and Immunogold Staining, we have analyzed the ultrastructure of platelet aggregates formed within the aperture and that are responsible for the occlusion. Standard electron microscopy showed that the aggregates formed on both collagen-epinephrine and collagenADP cartridges presented the same morphological features. The aggregates were exclusively composed of platelets, some of which were degranulated. Degranulation was particularly intense at the periphery of the aggregate where platelets were often totally devoid of secretory organelles. Immunogold Staining on ultrathin frozen sections with polyclonal antib...
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visualization of activation dependent epitopes on glycoprotein iib iiia complexes of platelets stimulated by thrombin Immunogold Staining of ultrathin sections
Seminars in Thrombosis and Hemostasis, 1996Co-Authors: M Humbert, Thomas J Kunicki, Claude Bihour, Joelle Winckler, Alan T Nurden, Paquita NurdenAbstract:: An immunoglobulin M monoclonal antibody (IgM MAb; AP-6) recognizing the sequence 211-221 of glycoprotein (GP) IIIa enabled a study of the distribution of this epitope on unstimulated or thrombin-activated platelets. Flow cytometry was used to evaluate the expression of this epitope on platelets and Immunogold Staining on ultrathin sections to analyze its distribution within the cell. There was little or no binding of AP-6 to unstimulated platelets, but Immunogold Staining showed labeling associated with the membranes of alpha-granules. The binding of AP-6 to thrombin-stimulated platelets was compared with that of anti-RIBS MAbs and antifibrinogen polyclonal antibodies. An increased expression of the AP6 epitope was observed on membranes of the surface-connected canalicular system and at the periphery of the cell as early as 10 to 15 seconds after platelet activation by thrombin. At the same time, binding of anti-RIBS MAbs confirmed that at least part of the endogenous fibrinogen had left the alpha-granules and was bound to platelet membranes. Staining with polyclonal antifibrinogen antibody also revealed fibrinogen in vesicles resulting from granule fusion. Rapidly, the pool of internal membranes with bound fibrinogen became exposed to the outside of the platelet, a process involving the unfolding of membranes and pseudopod formation. Our results provide evidence that activation of GP IIb-IIIa and binding of fibrinogen to platelet membranes can occur before their expression at the platelet surface. They also suggest the presence of an alpha-granule pool of GP IIb-IIIa linked to fibrinogen in unstimulated platelets.
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Glycoprotein Ia-IIa (VLA-2) and glycoprotein Ib-IX complexes are processed independently during thrombin-induced platelet activation.
Journal of Laboratory and Clinical Medicine, 1994Co-Authors: Paquita Nurden, Claude Bihour, Robert Combrié, Gralnick H, Alan T NurdenAbstract:We have previously shown that when human platelets are stimulated by thrombin, glycoprotein Ib-IX (GP Ib-IX) complexes are cleared to the surface-connected canalicular system (Blood 1990;76:1503). The question arose as to whether GP Ia-IIa complexes (VLA-2), another adhesion receptor thought to be linked to the membrane cytoskeleton, behaved similarly. Monoclonal antibodies to GP Ia-IIa were used in either (1) immunofluorescence procedures and flow cytometry or (2) Immunogold Staining and electron microscopy. In flow cytometry, VLA-2 was shown to have a low but variable expression in the platelets of different donors. Immunogold Staining showed that the complexes were regularly distributed over the platelet surface. This was best seen after Staining of paraformaldehyde-fixed whole mounts, where bound antibodies were often visualized in small clusters. The surface expression of VLA-2 receptors increased somewhat after thrombin stimulation, the receptors coming from a small intracellular pool revealed by flow cytometric analysis of Triton X-100-permeabilized cells. Immunogold Staining showed that after activation the receptors were equally as present on pseudopods as on the peripheral zone of the platelet. Down-regulation, as seen with GP Ib-IX complexes, was not observed. Our results therefore show that two-way trafficking of adhesion receptors can occur during platelet activation. This would imply that GP Ib-IX and VLA-2 are attached to different elements within the membrane cytoskeleton.
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Thrombin induces a rapid redistribution of glycoprotein Ib-IX complexes within the membrane systems of activated human platelets
Blood, 1990Co-Authors: P Hourdille, Robert Combrié, Eric Heilmann, Jglle Winckler, Kenneth J. Clemetson, Alan T NurdenAbstract:Previous studies have shown a decreased binding of monoclonal antibodies (MoAbs) to glycoprotein (GP) Ib-IX complexes on thrombin- stimulated platelets, but the reason for this is poorly understood. We have used (1) immunofluorescence procedures and flow cytometry, and (2) Immunogold Staining and electron microscopy to investigate this phenomenon. Washed platelets were incubated with alpha-thrombin, adenosine diphosphate, or ionophore A23187 for increasing lengths of time. For alpha-thrombin, but not the other agonists, flow cytometry confirmed a dose- and time-dependent decrease in the binding of MoAbs specific for GP Ib alpha (AP-1, Bx-1), GP IX (FMC 25), or to the complex itself (SZ 1). Immunoglold Staining performed using standard transmission or scanning electron microscopy high-lighted surface areas devoid of bound antibody. However, a quantitatively normal immunofluorescence was restored if paraformaldehyde-fixed, thrombin- stimulated platelets were permeabilized with Triton X-100 (Sigma Chemical Co, St Louis, MO) before MoAb addition, while Immunogold Staining was now seen to be concentrated within the interior of the platelet. Glutaraldehyde-fixed samples were then embedded in the resin Lowicryl K4M (Taab Laboratories Equipment Ltd, Aldermaston, England) and Immunogold Staining performed on thin sections using a polyclonal antibody to glycocalicin. An increased presence of GP Ib-IX complexes within surface-connected membrane systems of the thrombin-stimulated platelets was confirmed. Interestingly, GP Ib-IX movement was opposite to the thrombin-induced externalization of internal pools of GP IIb- IIIa complexes and of the alpha-granule membrane GP, GMP-140.
Paquita Nurden - One of the best experts on this subject based on the ideXlab platform.
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Ultrastructural analysis of the distribution of von Willebrand factor and fibrinogen in platelet aggregates formed in the PFA-100
Platelets, 1998Co-Authors: Christel Poujol, Alan T Nurden, Paponneau A, Eric Heilmann, Paquita NurdenAbstract:The PFA-100 is a new apparatus used to detect platelet dysfunction in vitro . Anticoagulated blood flows under constant pressure through a capillary, and across an aperture that pierces a membrane coated with collagen and either epinephrine or ADP. Through their ability to adhere and aggregate, platelets occlude the orifice and the closure time is a test of platelet function. Using electron microscopy and Immunogold Staining, we have analyzed the ultrastructure of platelet aggregates formed within the aperture and that are responsible for the occlusion. Standard electron microscopy showed that the aggregates formed on both collagen-epinephrine and collagenADP cartridges presented the same morphological features. The aggregates were exclusively composed of platelets, some of which were degranulated. Degranulation was particularly intense at the periphery of the aggregate where platelets were often totally devoid of secretory organelles. Immunogold Staining on ultrathin frozen sections with polyclonal antib...
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visualization of activation dependent epitopes on glycoprotein iib iiia complexes of platelets stimulated by thrombin Immunogold Staining of ultrathin sections
Seminars in Thrombosis and Hemostasis, 1996Co-Authors: M Humbert, Thomas J Kunicki, Claude Bihour, Joelle Winckler, Alan T Nurden, Paquita NurdenAbstract:: An immunoglobulin M monoclonal antibody (IgM MAb; AP-6) recognizing the sequence 211-221 of glycoprotein (GP) IIIa enabled a study of the distribution of this epitope on unstimulated or thrombin-activated platelets. Flow cytometry was used to evaluate the expression of this epitope on platelets and Immunogold Staining on ultrathin sections to analyze its distribution within the cell. There was little or no binding of AP-6 to unstimulated platelets, but Immunogold Staining showed labeling associated with the membranes of alpha-granules. The binding of AP-6 to thrombin-stimulated platelets was compared with that of anti-RIBS MAbs and antifibrinogen polyclonal antibodies. An increased expression of the AP6 epitope was observed on membranes of the surface-connected canalicular system and at the periphery of the cell as early as 10 to 15 seconds after platelet activation by thrombin. At the same time, binding of anti-RIBS MAbs confirmed that at least part of the endogenous fibrinogen had left the alpha-granules and was bound to platelet membranes. Staining with polyclonal antifibrinogen antibody also revealed fibrinogen in vesicles resulting from granule fusion. Rapidly, the pool of internal membranes with bound fibrinogen became exposed to the outside of the platelet, a process involving the unfolding of membranes and pseudopod formation. Our results provide evidence that activation of GP IIb-IIIa and binding of fibrinogen to platelet membranes can occur before their expression at the platelet surface. They also suggest the presence of an alpha-granule pool of GP IIb-IIIa linked to fibrinogen in unstimulated platelets.
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Glycoprotein Ia-IIa (VLA-2) and glycoprotein Ib-IX complexes are processed independently during thrombin-induced platelet activation.
Journal of Laboratory and Clinical Medicine, 1994Co-Authors: Paquita Nurden, Claude Bihour, Robert Combrié, Gralnick H, Alan T NurdenAbstract:We have previously shown that when human platelets are stimulated by thrombin, glycoprotein Ib-IX (GP Ib-IX) complexes are cleared to the surface-connected canalicular system (Blood 1990;76:1503). The question arose as to whether GP Ia-IIa complexes (VLA-2), another adhesion receptor thought to be linked to the membrane cytoskeleton, behaved similarly. Monoclonal antibodies to GP Ia-IIa were used in either (1) immunofluorescence procedures and flow cytometry or (2) Immunogold Staining and electron microscopy. In flow cytometry, VLA-2 was shown to have a low but variable expression in the platelets of different donors. Immunogold Staining showed that the complexes were regularly distributed over the platelet surface. This was best seen after Staining of paraformaldehyde-fixed whole mounts, where bound antibodies were often visualized in small clusters. The surface expression of VLA-2 receptors increased somewhat after thrombin stimulation, the receptors coming from a small intracellular pool revealed by flow cytometric analysis of Triton X-100-permeabilized cells. Immunogold Staining showed that after activation the receptors were equally as present on pseudopods as on the peripheral zone of the platelet. Down-regulation, as seen with GP Ib-IX complexes, was not observed. Our results therefore show that two-way trafficking of adhesion receptors can occur during platelet activation. This would imply that GP Ib-IX and VLA-2 are attached to different elements within the membrane cytoskeleton.
Janet M Powell - One of the best experts on this subject based on the ideXlab platform.
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Immunogold Staining of epoxy resin sections for transmission electron microscopy tem
CSH Protocols, 2008Co-Authors: Jeremy N Skepper, Janet M PowellAbstract:: INTRODUCTIONIn post-embedding methods of Immunogold Staining, the cells or tissues are fixed chemically or cryoimmobilized, dehydrated, and embedded in epoxy or acrylic resins. Thin sections (50-70 nm in thickness) are cut using an ultramicrotome with a diamond knife, using a water bath to collect the sections as they slide off the knife. The sections are stretched with solvent vapor or a heat source and collected onto either bare or plastic-coated nickel grids. The sections are then stained immunochemically with primary antibodies raised against antigens exposed on the surface of the sections. The primary antibodies are visualized by Staining immunochemically with secondary antibodies raised against the species and isotype of the primary antibodies, conjugated to colloidal gold particles. The immunochemically stained sections are then contrast stained with salts of uranium (uranyl acetate) and lead (lead citrate) to reveal the ultrastructure of the cells, and are finally viewed by transmission electron microscopy (TEM). Chemical fixation and embedding in a highly cross-linked epoxy resin is the method of choice for optimal ultrastructure and stability of the thin section in the electron beam. Immunogold Staining of thin epoxy resin sections, described here, is useful if the antigen of interest is very resistant to fixative, or if only archived material that was fixed primarily for ultrastructural studies is available.
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Immunogold Staining of london resin lr white sections for transmission electron microscopy tem
CSH Protocols, 2008Co-Authors: Jeremy N Skepper, Janet M PowellAbstract:: INTRODUCTIONIn post-embedding methods of Immunogold Staining, the cells or tissues are fixed chemically or cryoimmobilized, dehydrated, and embedded in epoxy or acrylic resins. Thin sections (50-70 nm in thickness) are cut using an ultramicrotome with a diamond knife, using a water bath to collect the sections as they slide off the knife. The sections are stretched with solvent vapor or a heat source and collected onto either bare or plastic-coated nickel grids. The sections are then stained immunochemically with primary antibodies raised against antigens exposed on the surface of the sections. The primary antibodies are visualized by Staining immunochemically with secondary antibodies raised against the species and isotype of the primary antibodies, conjugated to colloidal gold particles. The immunochemically stained sections are then contrast stained with salts of uranium (uranyl acetate) and lead (lead citrate) to reveal the ultrastructure of the cells, and are finally viewed by transmission electron microscopy (TEM). LR White was introduced as a low-toxicity alternative to epoxy resins, which frequently contained carcinogens. Unlike the simplest acrylic resins, in which monomers are polymerized to form long chains, the LR resins contain aromatic cross-linkers to improve the stability of the sections under the electron beam. LR White and Gold both have very low viscosity and readily penetrate, even into dense tissue. In this protocol, aldehyde-fixed tissue is dehydrated in ethanol, impregnated in LR White resin and polymerized under vacuum or in a nitrogen atmosphere before sectioning and Immunogold Staining.
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Immunogold Staining of ultrathin thawed cryosections for transmission electron microscopy tem
CSH Protocols, 2008Co-Authors: Jeremy N Skepper, Janet M PowellAbstract:: INTRODUCTIONA pre-embedding method of immunochemical Staining is used if antigens are damaged by resin embedding, or if the best preservation of membranes is required. Applying Immunogold reagents to sections of lightly fixed tissue, free of embedding medium, can be a very sensitive method of immunochemical Staining. Cells or tissues are fixed as strongly as possible and then treated with a cryoprotectant, which is usually a mixture of sucrose and polyvinylpyrrolidone (PVP). They are frozen onto pins in liquid nitrogen and sectioned at approximately -100°C. The frozen sections are thaw-mounted on to Formvar/nickel film grids and the cryoprotectant is removed by floating the grids on drops of phosphate-buffered saline (PBS). The Immunogold Staining is performed on the unembedded sections, which are subsequently contrast counterstained and infiltrated with a mixture of methylcellulose and uranyl acetate. In this protocol, samples are sectioned at low temperature, thaw-mounted onto film grids, immunochemically stained, contrast counterstained, and embedded/encapsulated in situ on the grid before viewing by transmission electron microscopy (TEM).
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Immunogold Staining following freeze substitution and low temperature embedding after chemical fixation or after cryoimmobilization for transmission electron microscopy tem
CSH Protocols, 2008Co-Authors: Jeremy N Skepper, Janet M PowellAbstract:: INTRODUCTIONIn this method for freeze substitution and low-temperature embedding in resin prior to Immunogold Staining, lightly fixed tissue pieces are cryoprotected by immersion in polypropylene glycol. The cryoprotected tissues are quench frozen and transferred under liquid nitrogen to vials containing frozen methanol or methanol containing uranyl acetate. The vials are transferred to a substitution vessel where the temperature can be controlled and a nitrogen atmosphere maintained. The temperature is raised (typically at 5°C/h to -90°C) and maintained for 24 h. This temperature is cold enough to prevent recrystallization of water and thus tissue disruption, but high enough for movement of water to occur and allow substitution with the liquid methanol. After 24 h, ~90% of the water has been substituted. The substitution medium is replaced and the temperature is raised to -70°C for 24 h. The substitution medium is changed again and the temperature is raised to -50°C. The tissue is impregnated with Lowicryl HM20 over a period of 1-5 d and the resin is polymerized by UV irradiation. Tissue is then sectioned and stained immunochemically with primary antibodies raised against antigens exposed on the surface of the sections, and primary antibodies are visualized by Staining with secondary antibodies conjugated to colloidal gold particles. The immunochemically stained sections are contrast stained with uranyl acetate and lead citrate to reveal the ultrastructure of the cells, and are finally viewed by transmission electron microscopy (TEM). This is the simplest and most versatile of the post-embedding procedures.
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Ultrastructural immunochemistry.
CSH protocols, 2008Co-Authors: Jeremy N Skepper, Janet M PowellAbstract:INTRODUCTIONThe use of colloidal gold technology was undoubtedly the most significant event in the development of immunochemistry. Gold particles are particularly useful for transmission electron microscopy (TEM) studies, because they scatter electrons strongly and even small particles are clearly visible under the electron microscope. Before proceeding to Immunogold Staining, it is important to gather as much information as possible about the antibody of interest and its respective antigen: Where is it likely to be located? Is the antigen extracellular, intracellular, membrane-associated, or a soluble component of the cytoplasm? Is it present in significant quantities? Is it sequestered at high concentration in any specific subcellular compartment, such as the mitochondria or the nucleus? How vulnerable to fixation and embedding is the antigen of interest? Information on the specificity of antibodies from Western blotting is valuable, but is not guaranteed to be useful for immunochemistry. Antibodies that "work well" on blots frequently have to be used at concentrations of up to three or more orders of magnitude greater for immunofluorescence and even more for Immunogold Staining studies, and some antibodies simply cannot be used for immunochemistry. This article describes methods and considerations for the use of Immunogold Staining, including fixation, controls, resolution, and quantification.
J H W M Rombout - One of the best experts on this subject based on the ideXlab platform.
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identification of two somatostatin immunoreactive cell types in the principal islet of sparus auratus l teleostei by Immunogold Staining
General and Comparative Endocrinology, 1990Co-Authors: M E Abad, M T Lozano, J J Tavernethiele, J H W M RomboutAbstract:Abstract Two types of somatostatin (SST 14)-immunoreactive cells are identified by Immunogold Staining in the Lowicryl-embedded principal islet of Sparus auratus: D1 cells, having large moderate to low electron dense granules, located between A cells in the islet periphery and D2 cells, containing smaller electron-dense granule s,.present between B cells in the central region of the islet. Although SST 28-like immunoreactivity was not observed in D cells of S. auratus, the presence of SST 14 and a SST 22-,25-, or 28-like sequence in D2 and D1 cells, respectively, is discussed. A third SST 14-immunoreactive cell, found in the islet periphery, showed immunoreactive D1- and unreactive A-like granules. This cell type, which has a pyknotic-like nucleus and a dark appearance in osmicated Epon-embedded tissue, is supposed to be the product of fusion of D1 and A cells.
Sheldon Penman - One of the best experts on this subject based on the ideXlab platform.
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Localization of heterogeneous nuclear ribonucleoprotein in the interphase nuclear matrix core filaments and on perichromosomal filaments at mitosis.
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: D C He, T. Martin, Sheldon PenmanAbstract:Although heterogeneous nuclear RNA (hnRNA) has been localized to the core filament substructure of the nuclear matrix, its precise location in the filament network has been unknown. The fA12 monoclonal antibody can localize, at high resolution, hn ribonucleoproteins (hnRNPs) and, presumably, hnRNA. Gold bead immunolabeling of resinless electron microscopy sections showed the fA12 antigens were in the fibrogranular material enmeshed in the filament network and not in the filaments themselves. At mitosis, hnRNP antigens became dispersed into a halo surrounding the chromosomes and spindle poles. Immunogold Staining showed fA12 stained fibrogranular material associated with perichromosomal and pericentriolar filaments distinct from the mitotic spindle fibers. fA12 also labeled the midbody remaining after cytokinesis.
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immunolocalization in three dimensions Immunogold Staining of cytoskeletal and nuclear matrix proteins in resinless electron microscopy sections
Proceedings of the National Academy of Sciences of the United States of America, 1990Co-Authors: Jeffrey A Nickerson, Gabriela Krockmalnic, Dacheng He, Sheldon PenmanAbstract:Abstract We describe two methods for Staining resinless thin sections with antibodies and gold-conjugated second antibodies. Immunolocalization of specific proteins is a powerful tool for cell structure studies but current techniques do not develop its full potential. Immunofluorescence provides only low-resolution localization, whereas conventional thin-section electron microscopy images and immunostains only the section surface. Resinless sections of extracted cell structures offer a simple and effective means of immuno-electron microscopy. Without embedding plastic or soluble proteins, the cell cytostructure produces high-contrast, three-dimensional images. Resinless sections of detergent-extracted cells are prepared by embedding in diethylene glycol distearate, sectioning, and removing diethylene glycol distearate before microscopy. In the first method of immunoStaining, extracted cells were fixed and stained with antibodies before embedment, sectioning, removal of the embedding resin, and critical point drying. In the postembedment method, the sample was embedded and sectioned, the diethylene glycol distearate was removed, and the sample was rehydrated before antibody Staining. With these techniques, specific proteins were localized with high resolution throughout the entire section. Stereoscopic micrographs of resinless sections revealed the precise localization of specific cytoskeleton and nuclear matrix proteins in three dimensions with unprecedented clarity.