In:
Microscopy and Microanalysis, Oxford University Press (OUP), Vol. 5, No. S2 ( 1999-08), p. 478-479
Abstract:
Fluorescein and the 1.4 nm Nanogold ® gold cluster label may be incorporated into a single Fab’ immunoprobe by separate cross-linking reactions, to give a probe which labels antigenic sites in a single step for correlative fluorescence and electron microscope visualization. These probes show high labeling density, labeling a pre-mRNA splicing factor in the HeLa cell nucleus; Microtubules were also densely labeled using fluorescence, other optical modalities, and electron microscopy; in a parallel experiment, a 5 nm colloidal gold probe gave only occasional labeling. We now describe Fab’ and streptavidin probes containing both Nanogold ® and the fluorescent cyanine dye, Cy3. F(ab’) 2 Goat anti-Mouse IgG and F(ab’) 2 goat anti-rabbit IgG fragments were reductively cleaved to Fab’ fragments using dithiothreitol (DTT) or mercaptoethylamine hydrochloride (MEA), which selectively reduce the F(ab’) 2 hinge disulfide bonds, with 5 mm EDTA to prevent reoxidation. Fab’ fragments were isolated by gel filtration (coarse gel: GH25, Amicon) then labeled with Monomaleimido- Nanogold ® which reacts site-specifically with thiols. Streptavidin was labeled using Mono- Sulfo -NHS-Nanogold ® at pH 7.5. Nanogold ® conjugates were isolated by gel filtration (Superose-12 column, Pharmacia), then reacted with excess Cy3 monofunctional NHS ester (labeling kit, Amersham Life Sciences) at pH 7.5; dual-labeled conjugates were isolated by gel filtration (Superose-12).
Type of Medium:
Online Resource
ISSN:
1431-9276
,
1435-8115
DOI:
10.1017/S1431927600015713
Language:
English
Publisher:
Oxford University Press (OUP)
Publication Date:
1999
detail.hit.zdb_id:
1481716-0
SSG:
11
SSG:
12
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