Lectin and immunolabeling of microvascular endothelia

Milici, Anthony J.; Porter, Glyn A.

doi:10.1002/jemt.1060190306

Cited by 12 publications

(3 citation statements)

References 33 publications

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“…After an additional incubation on ice for 30 min, the supernatant was aspirated, and the cell pellet was gently removed from the microcentrifuge tube and infiltrated with 50% polyvinylpyrrolidone in 0.1 M phosphate buffer, pH 7.4, containing 2.3 M sucrose overnight at 4°C. Thick 0.5-m cryosections of the pellets were prepared (21) and then incubated with the anti-gene 10 Ab or control mouse IgG (1:100) for 1 h followed by incubation with 5 g/ml Texas Red-conjugated donkey anti-mouse IgG. Exposure-matched fluorescence micrographs were taken on a Nikon FXA microscope.…”

Section: Assessment Of Levels Of Kv13 Expression In Cv-1 Cellsmentioning

confidence: 99%

Purification, Visualization, and Biophysical Characterization of Kv1.3 Tetramers

Spencer

Sokolov

et al. 1997

Journal of Biological Chemistry

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The voltage-gated K ؉ channel of T-lymphocytes, Kv1.3, was heterologously expressed in African Green Monkey kidney cells (CV-1) using a vaccinia virus/T7 hybrid expression system; each infected cell exhibited 10 4 to 5 ؋ 10 5 functional channels on the cell surface. The protein, solubilized with detergent (3-[cholamidopropyl)dimethylammonio]-1-propanesulfonic acid or cholate), was purified to near-homogeneity by a single nickel-chelate chromatography step. The Kv1.3 protein expressed in vaccinia virus-infected cells and its purified counterpart are both modified by a ϳ2-kDa coresugar moiety, most likely at a conserved N-glycosylation site in the external S1-S2 loop; absence of the sugar does not alter the biophysical properties of the channel nor does it affect expression levels. Purified Kv1.3 has an estimated size of ϳ64 kDa in denaturing SDS-polyacrylamide electrophoresis gels, consistent with its predicted size based on the amino acid sequence. By sucrose gradient sedimentation, purified Kv1.3 is seen primarily as a single peak with an approximate mass of 270 kDa, compatible with its being a homotetrameric complex of the ϳ64-kDa subunits. When reconstituted in the presence of lipid and visualized by negative-staining electron microscopy, the purified Kv1.3 protein forms small crystalline domains consisting of tetramers with dimensions of ϳ65 ؋ 65 Å. The center of each tetramer contains a stained depression which may represent the ion conduction pathway. Functional reconstitution of the Kv1.3 protein into lipid bilayers produces voltage-dependent K ؉ -selective currents that can be blocked by two high affinity peptide antagonists of Kv1.3, margatoxin and stichodactylatoxin.

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Section: Assessment Of Levels Of Kv13 Expression In Cv-1 Cellsmentioning

confidence: 99%

Purification, Visualization, and Biophysical Characterization of Kv1.3 Tetramers

Spencer

Sokolov

et al. 1997

Journal of Biological Chemistry

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“…A wide variety of lectins bind to the endothe lial surface and have been used, particularly in the mi crovasculature, to characterise differences in composition both between capillary beds and in microdomains on individual cells [17][18][19][20]. Our objective was to obtain a preliminary characterisation of variations on light micro scopic length scales, and we used fluorescently labelled wheat germ agglutinin (WGA) for this purpose.…”

Section: Introductionmentioning

confidence: 99%

Focal and Regional Variations in the Composition of the Glycocalyx of Large Vessel Endothelium

et al. 1994

J Vasc Res

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The glycocalyx of the endothelium of the systemic arteries and vena cava of the rabbit was visualised by in situ perfusion fixation with glutaraldehyde containing Alcian blue. The thickness of the layer ranged from 45 ± 1 nm in the coronary artery to 81 ± 2 nm in the carotid. The glycocalyx was 20 ± 1.5 nm thicker on the downstream side of intercostal ostia than on the upstream side. Changes in the staining pattern with increasing concentrations of MgCl₂ indicated that carboxyl groups made the major contribution to the surface charge, though sulphate groups were also present, particularly in the aortic arch and carotid artery. Segments of the thoracic aorta and carotid artery were also stained in vitro with fluorescence labelled wheat germ agglutinin, and fluorescence intensity in histological sections was quantified using a video microscope equipped with a microcomputer-based image analysis system. The fluorescence intensity in the carotid was 1.65 ± 0.15 times that in the aorta. Pretreatment with neuraminidase reduced fluorescence intensity by 60 ± 4% in the carotid and 53 ± 2% on the upstream side of intercostal ostia, but only by 37 ± 3% on the downstream side. Chondroitinase and heparanase both reduced binding and when used together their effect was additive, reducing fluorescence by 27 ± 3, 51 ± 4, and 32 ± 3% at the three sites, respectively. Though the interpretation of the lectin binding experiments is complicated by a number of factors, these results support previous reports that sialyl groups are abundant in the endothelial glycocalyx. Glycosaminoglycans are also present, however, in significant amounts.

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“…Living EC can be immunolabeled using peroxidase-conjugated antibodies (59,103) or with immunogold preparations (103,104) in direct or indirect procedures. As was mentioned in the explanation of the immunofluorescence technique, the cultures have to be kept at about 10°C to avoid phagocytosis of gold particles.…”

Section: The Demonstration Of Am By Using Electron Microscopymentioning

confidence: 99%

Basic Cell Culture Protocols

Pollard¹,

Walker²

1997

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Lectin and immunolabeling of microvascular endothelia

Cited by 12 publications

References 33 publications

Purification, Visualization, and Biophysical Characterization of Kv1.3 Tetramers

Purification, Visualization, and Biophysical Characterization of Kv1.3 Tetramers

Focal and Regional Variations in the Composition of the Glycocalyx of Large Vessel Endothelium

Basic Cell Culture Protocols

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