Cell surface labelling with gold colloid particulates: the use of avidin and staphylococcal protein A‐coated gold in conjunction with biotin and fc‐bearing ligands

Tolson, N.D.; Boothroyd, B.; Hopkins, Colin R.

doi:10.1111/j.1365-2818.1981.tb01296.x

Cited by 39 publications

(17 citation statements)

References 28 publications

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“…The high resolution immunolabelling technique we have used has resulted in the most detailed picture of the molecular organisation of the desmosome so far presented. A previous estimate of the resolution obtainable using this immunolabelling technique has suggested that the maximum possible distance between the gold particle observed and the antigen molecule it is labelling can be no greater than 12 nm (Tolson et al, 1981). We show that the major groups of desmosomal proteins and glycoproteins make distinctly different contributions to desmosomal structure.…”

Section: Discussionmentioning

confidence: 57%

Localisation of the protein and glycoprotein components of bovine nasal epithelial desmosomes by immunoelectron microscopy.

Miller¹,

Mattey²,

Measures³

et al. 1987

The EMBO Journal

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Desmosomal proteins (dp1‐4) and glycoproteins (dg1‐3) have been localised within desmosomes of bovine nasal epithelium by immunogold labelling of ultrathin frozen sections. Beginning in the extracellular space and proceeding through the plaque to the tonofilaments, the following localisations were found. Labelling for the 130,000 and 115,000 Mr glycoproteins (dg2 and dg3) was predominantly in the extracellular space, a location consistent with their proposed adhesive function. The glycoproteins of 175,000‐164,000 Mr (dg1) were also found in the extracellular space and in addition had cytoplasmic domains extending throughout the cytoplasmic plaque. The 83,000 Mr protein (dp3) was located along the cytoplasmic face of the membrane and extended into the plaque, whereas an antibody which recognises both the 83,000 Mr protein (dp3) and the 75,000 Mr protein (dp4) gave labelling both in and beyond the plaque. Labelling for the high mol. wt proteins of Mr 250,000 and 215,000 (dp1 and dp2) was largely excluded from the plaque, and was located distally, adjacent to the tonofilaments. Hemidesmosomes could not be labelled with antibodies to dg1‐3 or dp3 and 4, but some labelling was obtained with antibody to dp1 and 2.

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Section: Discussionmentioning

confidence: 57%

Localisation of the protein and glycoprotein components of bovine nasal epithelial desmosomes by immunoelectron microscopy.

Miller¹,

Mattey²,

Measures³

et al. 1987

The EMBO Journal

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“…sulphated glycoprotein C purified by SDS-gel electrophoresis from RM (Cooper et al, 1981 Vuento and Vaheri (1979 For immunoelectron microscopy, grids were incubated with appropriate antisera dilutions for 2 h at 40C and rinsed on 5% bovine serum albumin (BSA) in PBS. Grids were then incubated with protein A-gold complex (Tolson et al, 1981) for 1 h at 40C and rinsed again in BSA/PBS. For double labelling, sequential incubations in which the first antibody was followed by protein A-gold 12 rnm anid the second antibody was followed by protein A-gold 5 mn, were employed.…”

Section: Methodsmentioning

confidence: 99%

Localization of fibronectin, laminin-entactin, and entactin in Reichert's membrane by immunoelectron microscopy.

Semoff¹,

Hogan²,

Hopkins³

1982

The EMBO Journal

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Immunoelectron microscopy using protein A‐colloidal gold complexes of different sizes was used to study the relative distribution of extracellular matrix glycoproteins within Reichert's membrane (RM) of 13.5‐day mouse embryos. Labelling for fibronectin was distributed asymmetrically; the highest concentration occurring in the outermost layer adjacent to the trophoblast cells and negligible labelling in the inner matrix, beneath the parietal endoderm cells. Within the main body of the membrane, fibronectin was concentrated in discrete electron‐opaque deposits. Antibodies raised against the native complex between laminin and entactin, and against entactin alone labelled the RM more uniformly.

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“…Despite the apparent advantages of histochemical methods based on the interaction of avidin with biotin, this system is not used frequently for electron microscopy in conjunction with colloidal gold. Avidin-gold conjugates are not prepared easily and have a tendency towards high nonspecific binding (28,38); streptavidin-gold conjugates, however, are less prone to these problems. In addition, these gold probes appear to be less stable during storage than other gold reagents (such as antibody-gold or protein A-gold); the proteins dissociate from the gold particles leading to the instability of the colloid, the level of labeling is low, and there are difficulties in reciprocating the labeling.…”

Section: The Advantages Of the Sbbg Methodsmentioning

confidence: 99%

Uptake of maternal immunoglobulins in the enterocytes of suckling piglets: improved detection with a streptavidin-biotin bridge gold technique.

Kömüves¹,

Heath²

1992

J Histochem Cytochem.

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In ungulates, intestinal absorption of maternal immunoglobulins from colostrum plays a vital role in the acquisition of passive immunity during early neonatal life. In the present study we used post-embedding colloidal gold labeling to examine the intracellular localization of IgG in the jejunal enterocytes of miniature piglets suckled for 2 hr. Quantitation of the immunolabeling revealed that the most sensitive technique for IgG detection was the streptavidin bridge-gold technique. In this method, the LR White-embedded sections were labeled sequentially with biotinylated anti-porcine IgG, streptavidin, and biotinylated BSA conjugated to 10-nm colloidal gold. With this approach, we found the following sequence of maternal IgG accumulation: passage of IgG from colostrum through the brush border; binding to the apical plasma membrane; uptake in noncoated pits and invaginations; transport in endocytotic vesicles; and accumulation in granules in the apical cytoplasm.

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Cell surface labelling with gold colloid particulates: the use of avidin and staphylococcal protein A‐coated gold in conjunction with biotin and fc‐bearing ligands

Cited by 39 publications

References 28 publications

Localisation of the protein and glycoprotein components of bovine nasal epithelial desmosomes by immunoelectron microscopy.

Localisation of the protein and glycoprotein components of bovine nasal epithelial desmosomes by immunoelectron microscopy.

Localization of fibronectin, laminin-entactin, and entactin in Reichert's membrane by immunoelectron microscopy.

Uptake of maternal immunoglobulins in the enterocytes of suckling piglets: improved detection with a streptavidin-biotin bridge gold technique.

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