Staphylococcal protein a bound to colloidal gold: A useful reagent to label antigen-antibody sites in electron microscopy

Romano, Egidio; Romano, Mirtha

doi:10.1016/0019-2791(77)90146-x

Cited by 175 publications

(36 citation statements)

References 7 publications

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“…On the ultrastructural level, this method has been boosted by the development of colloidal gold-coupled protein A or secondary antibodies (Romano and Romano 1977;Romano et al 1974;Roth et al 1978), which allow to localize a speciWc antigenic site (for review see Roth 1983). Labeling eYciency depends on the speciWc antigen-antibody combination, the Wxation and embedding protocol (pre-embedding approaches are not considered).…”

Section: Immunolabeling Of High Pressure Frozen Samplesmentioning

confidence: 99%

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Studer

Humbel

Chiquet

2008

Histochem Cell Biol

250

192

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Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological "nanomachines" it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde Wxation, dehydration and staining, but also section thickness reduces it to some nanometers. CryoWxation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 m in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryoWxation methodology and compares its results with those of conventional procedures.Moreover, recent Wndings will be discussed showing that molecular models of proteins can be Wtted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.

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Section: Immunolabeling Of High Pressure Frozen Samplesmentioning

confidence: 99%

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Studer

Humbel

Chiquet

2008

Histochem Cell Biol

250

192

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“…Another promising group of particulate labels that came into the scope recently are gold colloids (8,10,16) . Gold particles can be obtained in different sizes and are pluripotent cytochemical markers because they can be associated with various "bridging" proteins such as antigens (22), lectins (15,16,32), immunoglobulins (8,29), and PA (1, 8,28,31) . For the immunocytochemical localization ofantigens, PA/ An probes have already been used as surface labels of resin sections (1, 31), but their usefulness on frozen sections to our knowledge has not yet been shown.…”

Section: Colloidal Gold Labeling Of Frozen Sectionsmentioning

confidence: 99%

Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections.

Geuze¹,

Slot²,

Ley³

et al. 1981

The Journal of Cell Biology

386

171

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Complexes of protein-A with 5 and 16 nm colloidal gold particles (PA/Au' and PA/ Au") are presented as sensitive and clean immunoprobes for ultrathin frozen sections of slightly fixed tissue . The probes are suitable for indirect labeling and offer the opportunity to mark multiple sites. The best procedure for double labeling was to use the smaller probe first, i .e ., antibody 1 -PA/Au' -antibody 2 -PA/Au' s. When this was done, no significant interference between PA/Au' and PA/Au 16 occurred . Using this double-labeling procedure we made an accurate comparison between the subcellular distributions of amylase as a typical secretory protein and of GP-2 a glycoprotein, characteristic for zymogen granule membrane (ZGM) preparations .We prepared two rabbit antibodies against GP-2 . One antibody (R x ZGM) was obtained by immunizing with native membrane material . The specificity of R x ZGM was achieved by adsorption with the zymogen granule content subfraction. The other, R x GP-2, was raised against the GP-2 band of the SDS polyacrylamide profile of ZGM. We found that the carbohydrate moiety of GP-2 was involved in the antigenic determinant for R x ZGM, while R x GP-2 was most likely directed against GP-2 polypeptide backbone .The immunocytochemical observations showed that GP-2, on the one hand, exhibited the characteristics of a membrane protein by its occurrence in the cell membrane, the Golgi membranes, and its association with the membranes of the zymogen granules . On the other hand, GP-2 was present in the contents of the zymogen granules and in the acinar and ductal lumina . Also, a GP-2-like glycoprotein was found in the cannulated pancreatic secretion (Scheffer et al ., 1980, Eur. J. Cell Biol. 23 :122-128) . Hence, GP-2 should be considered as a membrane-associated secretory protein of the rat pancreas .Ultrathin frozen sections of mildly fixed cells and tissues are very suitable for localizing intracellular antigens by means of immunoferritin cytochemistry (12,27,38). The major advantages of the technique are (a) the use of a particulate label, ensuring an accurate positioning of binding sites with high resolution, (b) a good accessibility of membrane-enclosed antigens. Recent developments (37) also allow excellent delineation of ultrastructural detail.As ferritin was the only suitable electron-dense label for frozen sections until now, a limitation was that only one antigen per section could be studied. Recently, however, iron-dextran particles have been introduced .

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“…In order to study the function of these and other proteins in the replication process it is important to know their location and time of appearance in the infected cell. Immunogold labelling, which is based on specific binding of Protein A-gold complexes to antigen antibody complexes (Romano & Romano, 1977), offers great potential for the h~ situ t Present address: Research Institute for Plant Protection, P.O. Box 9060, 6700 GW Wageningen, The Netherlands.…”

Section: Introductionmentioning

confidence: 99%

Immunogold Detection of Polyhedrin, p10 and Virion Antigens in Autographa californica Nuclear Polyhedrosis Virus-infected Spodoptera frugiperda Cells

Wilk¹,

Lent

Vlak

1987

Journal of General Virology

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SUMMARYProtein A-gold labelling was used to detect and locate particles and two major late antigens, polyhedrin and pl0, of Autographa californica multiply-enveloped nuclear polyhedrosis virus in thin sections of Spodoptera frugiperda cells. With polyhedrin antiserum, gold label was found over viral occlusion bodies (polyhedra), but not over the fibrous structures associated with polyhedron morphogenesis and previously thought to consist of precondensed polyhedrin. In contrast, with pl0 antiserum, gold label was found only over these fibrous structures in the nucleus and similar structures in the cytoplasm. This suggests that pl0 is a major constituent of the fibrous structures and may, therefore, play an important role in polyhedron morphogenesis.

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Staphylococcal protein a bound to colloidal gold: A useful reagent to label antigen-antibody sites in electron microscopy

Cited by 175 publications

References 7 publications

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections.

Immunogold Detection of Polyhedrin, p10 and Virion Antigens in Autographa californica Nuclear Polyhedrosis Virus-infected Spodoptera frugiperda Cells

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