Double labeling of cell surface antigens imaged with backscattered electrons

Heier, Hans Erik; Falleth, E.

doi:10.1002/jemt.1060060111

Cited by 8 publications

(5 citation statements)

References 5 publications

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“…Hence, to diminish steric hindrance and to allow safe discrimination between two probe sizes in double-labeling experiments, 20 and 40 nm probes are the only feasible sizes to use. The simultaneous detection of two antigenic sites on cells labeled with 20 and 40 nm gold probes visualized in the BE1 mode has been reported (Namork et al, 1987;Soligo et al, 1986?. Silver enhancement has, since it was introduced by Danscher (1981?, frequently been applied in light microscopy (LM) and transmission electron microscopy (TEM? to intensify gold labels Birrell and Hedberg, 1987;Danscher et al, 1987;Holgate et al, 1983;Lackie et al, 1985;Scopsi and Larsson, 1985).…”

Section: Introductionmentioning

confidence: 91%

“…The backscatter electron imaging (BEI) mode of the scanning electron microscope has proved very useful in the detection of colloidal gold probes labeling cell surface antigens. The method, first introduced by Trejdosiewicz et al in 1981, has found widespread application, sucessfully applied by many workers (Birrell and Hedberg, 1987;deHarven et al, 1984deHarven et al, , 1986deHarven et al, , 1987aGoode and Maugel, 1987;Heier et al, 1988;Hodges et al, 1987;Namork et al, 1986Namork et al, , 1987Nava et al, 1984;Soligo et al, 1985Soligo et al, , 1986Studer and Hermann, 1986;Walther et al, 1984;Walther and Muller, 1986). Few of the probe sizes commercially available (5-40 nm) are practical to use in scanning electron microscopy (SEMI, however, and the choice becomes even more limited when two probes are to be discriminated as in double-labeling of cells.…”

Section: Introductionmentioning

confidence: 99%

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Silver enhancement of gold probes (5–40 nm): Single and double labeling of antigenic sites on cell surfaces imaged with backscattered electrons

Heier¹

1989

J. Elec. Microsc. Tech.

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Silver enhancement of immunogold-labeled cells was carried out to increase the applicability of colloidal gold probes for visualization in the backscatter electron imaging (BEI) mode of a scanning electron microscope. Optimum conditions were established for single particle discrimination and differential counting of labeling density at low magnifications. Red blood cells double-labeled with 15 + 40 nm and 5 + 20 nm gold probes were silver-enhanced for 6 min and 20 min, respectively, at which times both pairs of labels increased to about 25 + 50 nm. The gold probes still appeared spherical after enhancement and were easily discriminated. Cells were also single-labeled with the above probes and enhanced accordingly. The present method enables visualization of individual particles of any probe size, labeling one, or simultaneously two, antigenic sites on cell surfaces. The silver enhancement procedure thereby allows cells to be labeled with small probes with increased labeling efficiency.

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Section: Introductionmentioning

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Silver enhancement of gold probes (5–40 nm): Single and double labeling of antigenic sites on cell surfaces imaged with backscattered electrons

Heier¹

1989

J. Elec. Microsc. Tech.

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“…For double labeling of molecules exposed on cell surfaces we have com pared the value of two different methods, one using two identifier antibodies of different classes (one IgG and one IgM) as recently demonstrated by Namork et al (1987), and another using two ligands of unrelated chemistry (anti-immunoglobulin and biotin-streptavidin complex), as initially rec ommended by Yamada and Sano (1985). In both cases, the gold probes used were of different diameters (15 and 40 nm).…”

Section: Doubling Labelingmentioning

confidence: 99%

Backscattered Electron Imaging of the Colloidal Gold Marker on Cell Surfaces

Harven

1989

Colloidal Gold

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“…BE1 is advantageous over the secondary electron imaging (SEI) in that the metal coating, needed for SEI, does not obscure the label either as a coating or as a competing element with high atomic number. The high backscatter electron (BE) yield of the gold particles thus offers high contrast, and smaller probes and a wider range of probe sizes can be applied, being ideal for double labeling (de Harven and Soligo, 1989;de Harven et al, 1990;Namork and Heier, 1989;Namork et al, 1987Namork et al, ,1988Namork, 1991;Pawley and Albrecht, 1988;Soligo et al, 1986). Indirect immunolabeling using primary antibodies of different immunoglobulin classes, species, or different ligands was always used in these studies to avoid cross reactivity.…”

Section: Introductionmentioning

confidence: 99%

Backscatter electron imaging of double immunogold labeled erythrocytes using two primary monoclonal IgM antibodies

Heier

1994

Microscopy Res & Technique

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The majority of mouse monoclonal antibodies reacting with blood group epitopes on erythrocytes are of the IgM class, have equal light chain type, and are available as culture supernatants only. To study the interrelationship of the blood group antigens, a method is presented which allows double labeling applying two unconjugated monoclonal antibodies of the same class and species. The method comprises two indirect, sequential labelings using mouse IgM anti-A and anti-H as primary antibodies and two goat anti-mouse IgM conjugated to 30 and 20 nm colloidal gold particles as secondary antibodies. After labeling for the first antigen, free binding sites on the primary antibody are blocked by incubation with an unconjugated goat anti-mouse antibody. The free anti-species on the secondary antibody, conjugated to 30 nm gold particles, are inactivated by silver enhancement. The silver enhancement also enlarges the gold particle for optimal discrimination between the two particle sizes, which are chosen accordingly. Semiquantitations of double labeled cells from subgroup A2 and A3 were found to be in good agreement with the counts of the corresponding single labelings as well as between experiments, irrespective of which of the two antibodies was applied in the first labeling sequence. The results were in accordance with a reciprocal but nonlinear relationship between the A and H antigens and suggest different affinities of the two antibodies for the epitopes in the subgroups investigated, indicating different biochemistry of the antigen determinants.

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Double labeling of cell surface antigens imaged with backscattered electrons

Cited by 8 publications

References 5 publications

Silver enhancement of gold probes (5–40 nm): Single and double labeling of antigenic sites on cell surfaces imaged with backscattered electrons

Silver enhancement of gold probes (5–40 nm): Single and double labeling of antigenic sites on cell surfaces imaged with backscattered electrons

Backscattered Electron Imaging of the Colloidal Gold Marker on Cell Surfaces

Backscatter electron imaging of double immunogold labeled erythrocytes using two primary monoclonal IgM antibodies

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