scholarly journals Hapten-Conjugated Antibodies and Visual Markers Used to Label Cell-Surface Antigens for Electron Microscopy: An Approach to Double Labeling

1972 ◽  
Vol 69 (12) ◽  
pp. 3732-3736 ◽  
Author(s):  
M. E. Lamm ◽  
G. C. Koo ◽  
C. W. Stackpole ◽  
U. Hammerling
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Surface Antigens ◽  
Label Cell ◽  
Cell Surface Antigens ◽  
Double Labeling ◽  
Visual Markers

scholarly journals New immunolatex spheres: visual markers of antigens on lymphocytes for scanning electron microscopy.

1975 ◽  
Vol 64 (1) ◽  
pp. 75-88 ◽  
Author(s):  
R S Molday ◽  
W J Dreyer ◽  
A Rembaum ◽  
S P Yen
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Cell Surface ◽  
Fluorescent Microscopy ◽  
Surface Antigens ◽  
Cell Surface Antigens ◽  
Aqueous Emulsion ◽  
Visual Markers ◽  
Latex Spheres ◽  
Scanning Electron

New immunochemical reagents consisting of antibodies bound to small latex spheres were used as visual markers for the detection and localization of cell surface antigens by scanning electron microscopy. Cross-linked latex spheres of various sizes from 300 to 3,4000 A in diameter were synthesized by aqueous emulsion copolymerization of methacrylate derivatives containing hydroxyl and carboxyl functional groups. Proteins and other molecules containing primary amino groups were covalently bonded to the acrylic spheres under a variety of mild conditions by the aqueous carbodiimide, cyanogen bromide, and glutaraldehyde methods. For use in the indirect immunochemical-labeling technique, goat antibodies directed against rabbit immunoglobulins were bonded to the spheres. These immunolatex reagents were shown to bind only to cells (red blood and lymphocytes) which had previously been sensitized with rabbit antibodies against cell surface antigens. Mouse spleen lymphocytes with exposed immunoglobulins on their surface (B cells) were labeled with these spheres and distinguished from unlabeled or T lymphocytes by scanning electron microscopy. The distribution of Ig receptors on lymphocytes was also studied using the spheres as visual markers. When lymphocytes were fixed with glutaraldehyde and subsequently labeled with the immunolatex reagents, a random distribution was observed by scanning electron microscopy; a patchy distribution was observed when unfixed lymphocytes were used. These results are consistent with studies using ferritin-labeled antibodies (S. De Petris and M. Raff. 1973. Nature [Lond.]. 241:257.) and support the view that Ig receptors on lymphocytes undergo translational diffusion. In addition to serving as visual markers for scanning electron microscopy, these latex spheres tagged with fluorescent or radioactive molecules have applications as highly sensitive markers for fluorescent microscopy and as reagents for quantitative studies of cell surface antigens and other receptors.

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.

scholarly journals USE OF HYBRID ANTIBODY WITH ANTI-γG AND ANTI-FERRITIN SPECIFICITIES IN LOCATING CELL SURFACE ANTIGENS BY ELECTRON MICROSCOPY

1968 ◽  
Vol 128 (6) ◽  
pp. 1461-1473 ◽  
Author(s):  
Ulrich Hämmerling ◽  
Tadao Aoki ◽  
Etienne de Harven ◽  
Edward A. Boyse ◽  
Lloyd J. Old
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Cell Surface Antigens ◽  
Viable Cells ◽  
Dual Specificity ◽  
Visual Markers ◽  
A Cell ◽  
The Many ◽  
Hybrid Antibody ◽  
Mouse Antibody

Hybrid antibody [F(ab')2] with dual specificity for mouse γG and ferritin was prepared from the corresponding rabbit antisera, providing a precise reagent for locating mouse γG on cell surfaces. Viable cells were exposed successively to (a) mouse antibody to a cell surface antigen, (b) the rabbit hybrid antibody, and (c) ferritin, before preparation for electron microscopy. This method of See PDF for Structure. labeling is sensitive and specific and clearly lends itself to the introduction of visual markers other than ferritin. Other advantages are uniformity of labeling, ease of purification of the reagent, and circumvention of the many drawbacks arising from coupling ferritin to antibody chemically.

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