Detection of Small (5-15 nm) Gold-Labelled Surface Antigens by Back- Scattered Electrons

1985 ◽  
Vol 43 ◽  
pp. 538-541
Author(s):  
Paul Walther ◽  
Martin Müller
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Colloidal Gold ◽  
Three Dimensional ◽  
Surface Antigens ◽  
Uniform Size ◽  
Backscattered Electrons ◽  
Additional Information ◽  
Secondary Electron Mode ◽  
Electron Mode

In contrast to thin section transmission electron microscopy, scanning electron microscopy (SEM) permits “three dimensional” analysis of events occurring at the cell surface. Since it is now generally recognized that many important cellular functions are initiated and regulated via interactions taking place at the cell surface, SEM studies of surface antigens have become an area with increasing applications. Surface antigens have been visualized using antibody coupled phages, latex spheres, colloidal gold, i.e. markers that are recognized in the SEM either by their specific shape or because of their uniform size. An ideal marker, however, should be as small as possible to reduce the problems of sterical hinderance. All known markers smaller than approx. 15 nm (colloidal gold, ferritin) are round particles which in the secondary electron mode (SE) are hardly discerned from surface structures or contaminants of similar size. Therefore the markers must be amenable to verification using additional information such as backscattered electrons (BSE).

Immunogold staining method for the light microscopic detection of leukocyte cell surface antigens with monoclonal antibodies: its application to the enumeration of lymphocyte subpopulations.

1983 ◽  
Vol 31 (3) ◽  
pp. 376-381 ◽  
Author(s):  
M De Waele ◽  
J De Mey ◽  
M Moeremans ◽  
M De Brabander ◽  
B Van Camp
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Peripheral Blood ◽  
Colloidal Gold ◽  
Staining Method ◽  
Surface Antigens ◽  
Lymphocyte Subpopulations ◽  
Cell Surface Antigens ◽  
Immunogold Staining ◽  
Microscopic Detection

Colloidal gold was used as a marker for the light microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies. Suspensions of peripheral blood leukocytes were first incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. The cells were fixed and cytocentrifuge preparations or smears were made. Granulocytes and monocytes were then labeled by the cytochemical staining of their endogenous peroxidase activity. Lymphocytes reacting with the monoclonal antibody had numerous dark granules around the surface membrane. With electron microscopy, these granules appeared as patches of gold particles. This immunogold staining method proved to be a reliable tool for the enumeration of T-lymphocyte subpopulations in peripheral blood. The results were almost identical to those obtained with immunofluorescence microscopy. The procedure can also be applied on small volumes of capillary blood. This constitutes a good microtechnique for the determination of lymphocyte subsets in children.

Double labelling of cell surface antigens with colloidal gold markers

1990 ◽  
Vol 22 (1) ◽  
pp. 18-23 ◽  
Author(s):  
E. De Harven ◽  
D. Soligo ◽  
H. Christensen
Keyword(s):  
Cell Surface ◽  
Colloidal Gold ◽  
Surface Antigens ◽  
Double Labelling ◽  
Cell Surface Antigens

scholarly journals HAPTEN-SANDWICH LABELING

1974 ◽  
Vol 140 (2) ◽  
pp. 523-537 ◽  
Author(s):  
L. Wofsy ◽  
P. C. Baker ◽  
K. Thompson ◽  
J. Goodman ◽  
J. Kimura ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Surface Antigens ◽  
Cell Populations ◽  
Molecular Weight Protein ◽  
Protein Markers ◽  
High Molecular Weight Protein ◽  
Cell Surface Antigens ◽  
A Cell ◽  
Weight Protein

A hapten-sandwich procedure has been developed for specific labeling of cell surface antigens for fluorescence or electron microscopy. Haptens are azo-coupled to immunoglobulins specific for a cell surface antigen; the hapten-modified cell-bound antibodies can then be visualized by adding fluorescent antihapten antibody, or by adding antihapten antibody followed by hapten-modified markers for electron microscopy. Virus or high molecular weight protein markers are lightly cross-linked before conjugation with hapten to prevent their disruption. Such stable hapten-modified markers, and the accessibility of many different purified anti-azophenyl-hapten antibodies, make it feasible to distinguish more than one membrane antigen in a given labeling experiment. When mouse lymphoid cell populations are labeled with separate markers for Ig and for thymus-associated antigens, many cells exhibit the Ig marker exclusively or the thymic marker predominantly, and some cells are completely free of label.

scholarly journals Derivatized silica spheres as immunospecific markers for high resolution labeling in electron microscopy.

1978 ◽  
Vol 78 (2) ◽  
pp. 309-318 ◽  
Author(s):  
K R Peters ◽  
G Rutter ◽  
H H Gschwender ◽  
W Haller
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Cell Surface ◽  
Hela Cells ◽  
Average Diameter ◽  
Surface Antigens ◽  
Silica Sphere ◽  
Silica Spheres ◽  
Cell Surface Antigens ◽  
Controlled Pore Glass

For high resolution labeling of influenza virus cell surface antigens on HeLa cells, an immunospecific marker is used with silica sphere cores of 13--14 nm average diameter. These markers are formed using commercially available silica sphere sols. Two other size ranges are available, 7--8 nm and 22--25 nm. The steps for chemical derivatization are described in detail. Amino and aldehyde functions are covalently introduced onto the sphere surface. Sols of these derivatized silica spheres (DSS) are physicochemically stable and therefore usable for years. Coupling of IgG to DSS followed by permeation chromatography on controlled pore glass results in size-defined immunospecific silica sphere markers (DSS-markers). Saturation labeling of cell surface antigens on HeLa cells on cover slips is obtained with the final sphere concentration of 10(14) DSS-marker/cm3 within 20 min. With usual protective conditions, the marker stability and labeling ability are preserved for months. The visibility and the fine structure of the DSS-marker on cell surfaces are shown by using transmission electron microscopy (TEM) with stereo replicas and ultrathin sections.

scholarly journals Hapten-sandwich labeling. II. Immunospecific attachment of cell surface markers suitable for scanning electron microscopy.

1975 ◽  
Vol 64 (2) ◽  
pp. 311-321 ◽  
Author(s):  
M K Nemanic ◽  
D P Carter ◽  
D R Pitelka ◽  
L Wofsy
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Cell Surface ◽  
Mammary Tumor ◽  
Surface Antigens ◽  
Membrane Structures ◽  
Cell Surface Antigens ◽  
Mammary Tumor Cells ◽  
Tumor Virus ◽  
Scanning Electron

A hapten-sandwich procedure has been used for immunospecific labeling of cell surface antigens with markers visible by scanning electron microscopy. Antihapten antibody was used to link hapten-modified tobacco mosaic virus, bushy stunt virus, or hemocyanin to hapten-modified human erythrocytes. The antihapten antibody bridge was also used to link the hapten-virus marker to hapten-modified antibodies against mammary tumor virus on mouse mammary tumor cells, or against immunoglobulin receptors on mouse splenic lymphocytes. In all cases, labeling was highly specific. With this technique, it is possible to (a) compare morphological features of cells bearing differing cell surface antigens, and (b) examine the arrangement of specific antigenic sites on a cell surface or their distribution relative to membrane structures such as microvilli.

Colloidal Gold Markers: Preparation and Cell Labeling for Transmission and Scanning Electron Microscopy

1985 ◽  
Vol 43 ◽  
pp. 530-533
Author(s):  
Robert S. Molday
Keyword(s):  
Cell Surface ◽  
Colloidal Gold ◽  
Critical Evaluation ◽  
Cell Labeling ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Gold Particles ◽  
Backscattered Electrons ◽  
Wide Range ◽  
Scanning Electron

Colloidal gold particles have become one of the most widely used markers to detect, localize and, in some cases, quantitate cell surface and intracellular antigens and receptors since their introduction as transmission electron microscopic (TFM) markers by Faulk and Taylor in 1971 and as scanning electron microscopic (SEM) markers by Horisberger et al. in 1975. This interest in colloidal gold markers for cell labeling is based on their versatile properties for detection under the electron microscope. Colloidal gold particles are highly electron-dense which enables them to be seen under the TEM in thin sections of heavily stained cells. They can be prepared in a wide range of highly uniform sizes for visualization at different magnifications and for multiple labeling studies. Under the SEM, gold particles emit a high quantity of secondary electrons, backscattered electrons and characteristic X-ray signals and as a result, with the appropriate detectors, they can be readily distinguished from cell surface structures having a similar morphological appearance. The successful application of colloidal gold particles as markers for TEM and SEM however requires (i) careful preparation and characterization of both the gold markers and the ligand (protein)-gold conjugates, (ii) utilization of specific labeling techniques employing necessary controls to confirm the specificity of labeling, and (iii) critical evaluation of results in relation to the conditions used in labeling. These aspects of gold labeling will be considered here. Additional information can be obtained from recent reviews dealing specifically with gold markers and more generally with cell labeling techniques.

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