Immunogold Labeling for the Single-Laser FACS Analysis of Triple Antibody-Binding Cells

Taking advantage of the high elemental contrast of particles of colloidal gold observed in the backscattered electron imaging(BEI) mode of the SEM (1,2), the human T lymphocyte was chosen as a model system to study the potential value of immunogold labeling for the quantification of cell surface expressed molecules. The CD3 antigen which is expressed on all human T lymphocytes and is readily identified by the LEU-4 murine monoclonal antibody (Becton Dickinson, Mountain View, CA) followed by a gold conjugated goat anti-mouse Ig polyclonal antibody was chosen as a model target antigen. When quantified by non-EM methods, using radio-iodinated probes or FACS analysis, approximately 30,000 to 50,000 copies of this antigen per cell are enumerated.The following observations were made while attempting to quantify the same molecule by SEM after specific immunogold labeling:Imaging in the SE vs BE mode: The numbers of gold markers counted in the secondary electron (SE) imaging mode are considerably lower than those counted on the same cells in the backscattered electron (BE) imaging mode.

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Detection of triple antibody-binding lymphocytes in standard single laser flow cytometry using colloidal gold, fluorescein and phycoerythrin as labels

Journal of Immunological Methods ◽

10.1016/0022-1759(87)90211-0 ◽

1987 ◽

Vol 101 (1) ◽

pp. 23-28 ◽

Cited By ~ 25

Author(s):

Roger Festin ◽

Birgitta Björklund ◽

Thomas H. Tötterman

Keyword(s):

Flow Cytometry ◽

Colloidal Gold ◽

Antibody Binding ◽

Single Laser

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Structural organization of escherichia coli ribosomes as determined by immuno electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081693 ◽

1978 ◽

Vol 36 (2) ◽

pp. 166-167 ◽

Cited By ~ 1

Author(s):

G. Stöffler ◽

R.W. Bald ◽

J. Dieckhoff ◽

H. Eckhard ◽

R. Lührmann ◽

...

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Ribosomal Proteins ◽

Structural Organization ◽

Three Dimensional ◽

Ribosomal Subunit ◽

Spatial Arrangement ◽

Small Subunit ◽

Antibody Binding ◽

Immuno Electron Microscopy

A central step towards an understanding of the structure and function of the Escherichia coli ribosome, a large multicomponent assembly, is the elucidation of the spatial arrangement of its 54 proteins and its three rRNA molecules. The structural organization of ribosomal components has been investigated by a number of experimental approaches. Specific antibodies directed against each of the 54 ribosomal proteins of Escherichia coli have been performed to examine antibody-subunit complexes by electron microscopy. The position of the bound antibody, specific for a particular protein, can be determined; it indicates the location of the corresponding protein on the ribosomal surface.The three-dimensional distribution of each of the 21 small subunit proteins on the ribosomal surface has been determined by immuno electron microscopy: the 21 proteins have been found exposed with altogether 43 antibody binding sites. Each one of 12 proteins showed antibody binding at remote positions on the subunit surface, indicating highly extended conformations of the proteins concerned within the 30S ribosomal subunit; the remaining proteins are, however, not necessarily globular in shape (Fig. 1).

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Immunogold labeling of Pneumocystis carinii for Electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100085654 ◽

1991 ◽

Vol 49 ◽

pp. 270-271

Author(s):

Michael P. Goheen ◽

Marilyn S. Bartlett ◽

James W. Smith

Keyword(s):

Electron Microscopy ◽

Pneumocystis Carinii ◽

Severe Pneumonia ◽

Culture Fluid ◽

Immunogold Labeling ◽

Antigenic Sites ◽

Transmission Electron ◽

Response To Chemotherapy ◽

Acquired Immunodeficiency ◽

Immunodeficiency Syndrome

Studies of the biology of Pneumocystis carinii (PC) are of increasing importance because this extracellular pathogen is a frequent source of severe pneumonia in patients with acquired immunodeficiency syndrome (AIDS) and is a leading cause of mortality in these patients. Immunoelectron microscopic localization of antigenic sites on the surface of PC would improve the understanding of these sites and their role in pathenogenisis of the disease and response to chemotherapy. The purpose of this study was to develop a methodology for visualizing immunoreactive sites on PC with transmission electron microscopy (TEM) using immunogold labeled probes.Trophozoites of PC were added to spinner flask cultures and allowed to grow for 7 days, then aliquots of tissue culture fluid were centrifuged at 12,000 RPM for 30 sec. Pellets of organisims were fixed in either 1% glutaraldehyde, 0.1% glutaraldehyde-4% paraformaldehyde, or 4% paraformaldehyde for 4h. All fixatives were buffered with 0.1M Na cacodylate and the pH adjusted to 7.1. After fixation the pellets were rinsed in 0.1M Na cacodylate (3X), dehydrated with ethanol, and immersed in a 1:1 mixture of 95% ethanol and LR White resin.

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Glucose metabolism in cancer cells: immunogold localization of hexokinase to the outer mitochondrial membrane

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141597 ◽

1995 ◽

Vol 53 ◽

pp. 1044-1045

Author(s):

Krishan K. Arora ◽

Glenn L. Decker ◽

Peter L. Pedersen

Keyword(s):

Tumor Cells ◽

Mitochondrial Membrane ◽

Present Report ◽

Large Fraction ◽

Mitochondrial Fraction ◽

Immunogold Labeling ◽

Outer Mitochondrial Membrane ◽

Immunogold Localization ◽

Hexokinase Activity ◽

Normal Tissues

Hexokinase (ATP: D-hexose 6-phophotransferase EC 2.7.1.1) is the first enzyme of the glycolytic pathway which commits glucose to catabolism by catalyzing the phosphorylation of glucose with ATP. Previous studies have shown diat hexokinase activity is markedly elevated in rapidly growing tumor cells exhibiting high glucose catabolic rates. A large fraction (50-80%) of this enzyme activity is bound to the mitochondrial fraction (1,2) where it has preferred access to ATP (3). In contrast,the hexokinase activity of normal tissues is quite low, with one exception being brain which is a glucose-utilizing tissue (4). Biochemical evidence involving rigorous subfractionation studies have revealed striking differences between the subcellular distribution of hexokinase in normal and tumor cells [See review by Arora et al (4)].In the present report, we have utilized immunogold labeling techniques to evaluate die subcellular localization of hexokinase in highly glycolytic AS-30D hepatoma cells and in the tissue of its origin, i.e., rat liver.

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