Scanning electron microscopy of cell surface antigens labeled with colloidal gold

Harven, Étienne de; Soligo, Davide

doi:10.1002/aja.1001750212

Cited by 68 publications

(24 citation statements)

References 24 publications

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“…This prefix procedure was employed to prevent receptor endocytosis and loss of surface structure (de Harven, 1987;de Harven and Soligo, 1986). Thereafter, the slide was placed for 5 rnin in 0.1% glycine in PBS and then bathed twice for 5 rnin in PBS containing serum proteins to block nonspecific adsorption of the primary and/or secondary antibodies.…”

Section: Methodsmentioning

confidence: 99%

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Scanning electron microscopic study of immunogold‐labeled human leukocytes

Helinski

Bootsma

McGroarty

et al. 1990

J. Elec. Microsc. Tech.

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For many years critical point drying (CPD) has been the method of choice for preparing cells for scanning electron microscopy (SEM). Described herein is a simple, efficient, inexpensive, reproducible, and safe procedure using Peldri II, a proprietary fluorocarbon compound that is solid at room temperature and a liquid above 25 degrees C, as a sublimation dehydrant for processing specimens for SEM. The utility of Peldri II was demonstrated in studies using leukocytes from the blood of healthy donors and patients with leukemia as well as from long-term lymphoblastoid cell lines. The application of the proposed Peldri II procedure was further documented in SEM studies in which the expression and distribution of the interleukin-2 receptor (IL-2R) on leukocyte surface membranes was imaged using colloidal gold-labeled antibodies (i.e., immunogold). When compared with current SEM preparation procedures using CPD, Peldri II is a useful alternative that is thought to offer several important advantages.

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Section: Methodsmentioning

confidence: 99%

“…Secondary electron image (SEI) mode, backscatter electron image (BEI) mode, and mixed image (SEI/ BEI) mode were used to examine immunogold-labeled cells (de Harven et al, 1984;de Harven and Soligo, 1986;de Harven, 1987).…”

Section: Methodsmentioning

confidence: 99%

Scanning electron microscopic study of immunogold‐labeled human leukocytes

Helinski

Bootsma

McGroarty

et al. 1990

J. Elec. Microsc. Tech.

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“…We have studied the binding of such an antibody on A,, A,, A,, A, and A,, erythrocytes, using the backscattered electron imaging (BEI) mode of a scanning electron microscope. Bound antibody was revealed by anti-antibody conjugated to colloidal gold particles, which can be conveniently visualized because of their intense backscatter electron signal (11,12,13). Preliminary results of this study have been reported elsewhere (14).…”

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confidence: 90%

A study of immunogold‐labelled blood group A erythrocytes in the scanning electron microscope

Heier¹,

Namork²,

Falleth³

1988

European J of Haematology

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This report presents binding patterns on A1, A2, A3, Ax and Ae1 erythrocytes of a monoclonal anti‐A (A 003) antibody which reacts predominantly with difucosylated A oligosacccharides, visualized with colloidal gold particles in the backscattered electron imaging mode of a scanning electron microscope. A relatively weak labelling was found on most A, cells, while the labelling in subgroups A2, A3 and especially Ax appeared relatively stronger. Very few Ae1 cells were labelled. The results emphasize the qualitative uniqueness of A1 and suggest that many Ax cells have high proportions of difucosylated A oligosaccharides. Labelling variations were found between different A3 and Ax traits, and in all subgroups between cells and even between different parts of the cells. No immunolabelling differences were found in relation to secretor status.

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“…The cells in the negative control wells were incubated with DMEM and 10 mg . [25] was followed. The cells, grown on coverslips or micropore membranes were rinsed twice in 0.01 M PBS and lightly "prefixed" for 10 min in freshly prepared 0.2% glutaraldehyde solution with 4% sucrose buffered in 0.01 M PBS (pH 7.2).…”

Section: Enzyme-linked Immunosorbent Assaymentioning

confidence: 99%

Adrenalectomy permits a late, local TNF-α release in LPS-challenged rat airways

Miller-Larsson¹,

Runström²,

Brattsand³

1999

Eur Respir J

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The aim of this study was to investigate the variation of intercellular adhesion molecule (ICAM)-1 which occurs between individual airway epithelial cells of distinct phenotype. 16HBE14o ± (16HBE) and BEAS-2B cell lines and human airway explants were cultured with medium alone or a mixture of tumour necrosis factor (TNF)-a (10 ng . mL -1 ) and interferon (IFN)-c (40 ng . mL -1 ) before being immunogold-labelled and examined quantitatively using sensitive high-resolution electron microscopic techniques.By enzyme-linked immunosorbent assay there was a 1.6-fold increase of ICAM-1 in the BEAS-2B cells following the cytokine mix which was not apparent in the 16HBE cells. However, high-resolution scanning electron microscopy demonstrated that an upregulation had occurred; median and ranges for gold particle number per 10 mm 2 cell surface were: 7.9 (0±40) for nonstimulated and 19.1 (0±60 for stimulated) (p<0.01, Mann±Whitney U-test). The value for the nonstimulated BEAS-2B cells was 24.2 (0± 60), 3-times higher that the constituitive expression in the 16HBE cells (p<0.01), whereas following stimulation, it was 68.5 (20±130) (p<0.01). Values for explant epithelial outgrowths were similar to the 16HBE cells. Immunohistochemistry of the explanted mucosa showed both constitutive and upregulated expression of epithelial ICAM-1 associated with basal and indeterminate cells rather than with ciliated or goblet cells.These results using high-resolution techniques indicate that there is marked cell-tocell variation in cellular adhesion molecule expression and that it is the basal cells and less well differentiated (indeterminate) epithelial cells which are likely to play key roles in leukocyte retention via intercellular adhesion molecule-1. Eur Respir J 1999; 13: 1318±1328. Whilst ICAM-1 cell surface expression and alteration following stimulation of cultured cells has been determined by enzyme-linked immunoabsorbent assay (ELISA) and flow cytometry [4, 14±17], its cell surface localization, cell-to-cell variation and variation between cells of distinct phenotype can best be ascertained and quantified using high-resolution and immunohistological techniques [18]. These data are important as the ELISA technique measures a signal which is as a necessity averaged across the cell cultures and takes no account of the cell-to-cell variation nor the likelihood that cells of distinct phenotype may show large variations in their capacity to express ICAM-1.In the present study, two well-characterized human bronchial epithelial cell lines were used: 16HBE14o ± (16HBE) and BEAS-2B cells, and the results compared with primary cell outgrowths and intact mucosa in which distinct epithelial phenotypes could be identified. Whilst the expression of ICAM-1 on BEAS-2B cells has previously been reported by ELISA, the expression on 16HBE cells has not. The cells were stimulated in culture with a mixture of TNFa and IFN-c and the cell surface expression of ICAM-1 in stimulated and nonstimulated cells compared using two Eur Respir J 1999; 13...

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Scanning electron microscopy of cell surface antigens labeled with colloidal gold

Cited by 68 publications

References 24 publications

Scanning electron microscopic study of immunogold‐labeled human leukocytes

Scanning electron microscopic study of immunogold‐labeled human leukocytes

A study of immunogold‐labelled blood group A erythrocytes in the scanning electron microscope

Adrenalectomy permits a late, local TNF-α release in LPS-challenged rat airways

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