Immunoelectron microscopy in kidney research: Some contributions and limitations

Kerjaschki, Dontscho; Sawada, Hajime; Farquhar, Marilyn G.

doi:10.1038/ki.1986.175

Cited by 44 publications

(14 citation statements)

References 70 publications

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“…it displays some advantages also in comparison to other approaches. In fact, it possesses dominant sensitivity in comparison to postembedding IG labeling of cell surface-associated antigens, where the immunocytochemical access is restricted to the molecules exposed on the surface of sec tion [12,13]. Further, unlike most postembedding IG labelings, the preembedding IGS staining allows postfixa tion with osmium tetroxidc which well preserves mem brane profiles.…”

Section: Discussionmentioning

confidence: 99%

The Immunogold-Silver Staining Procedure in the Study of Freshly Suspended Langerhans Cells at the Transmission Electron Microscopic Level

et al. 1991

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The potential of an immunogold-silver staining for the study of human suspended Langerhans cells at the transmission electron microscopic level was evaluated. Cells were labeled, by using a preembedding technique, with 5-nm colloidal gold particles followed by silver enhancement. The use of small colloidal gold particles permits a detection of small quantities of antigen; the metallic silver deposition around gold granules gives rise to a large electron-dense marker which can be easily detected even at low magnification. Ultrastructural details were well preserved, and the background was not significant. The major advantage of the present immunogold-silver staining is that it enables to detect labeled cells easily, even when limited amounts of antigenic moieties are present on a low percentage of cells. Therefore, a rapid and simultaneous evaluation of both immunophenotype and ultrastructural details of investigated cells is allowed.

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Section: Discussionmentioning

confidence: 99%

The Immunogold-Silver Staining Procedure in the Study of Freshly Suspended Langerhans Cells at the Transmission Electron Microscopic Level

et al. 1991

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“…By and in the lamina fibroreticularis (region of the base-analogy to cartilage proteoglycan where a terminal sidement membrane below the lamina densa). Such an ori-chain-free region of the core protein is involved in linkentation could theoretically give rise to immunolabeling age to hyaluronic acid (reviewed by , the of the lamina lucida (Stow et al, 1985;Kerjaschki et al, side-chain-free regions of the large proteoglycan may 1986) with an antibody whose specificity was confined play a similarly important role in anchorage to moleto the region of the core protein containing side chains. cules of the basement membrane, such as type IV collaThe large proteoglycan has specific binding sites on type gen and laminin.…”

Section: Location Of the Large Heparan Sulfate Proteoglycan In Basemementioning

confidence: 98%

Visualization of the large heparan sulfate proteoglycan from basement membrane

et al. 1988

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Kleinschmidt spreading, negative staining, and rotary shadowing were used to examine the large form of (basement membrane) heparan sulfate proteoglycan in the electron microscope. Heparan sulfate proteoglycan was visualized as consisting of two parts: the core protein and, emerging from one end of the core protein, the glycosaminoglycan side chains. The core protein usually appeared as an S-shaped rod with about six globules along its length. Similar characteristics were observed in preparations of core protein in which the side chains had been removed by heparitinase treatment ("400-kDa core") as well as in a 200-kDa trypsin fragment ("P200") derived from one end of the core protein. The core protein was sensitive to lyophilization and apparently also to the method of examination, being condensed following Kleinschmidt spreading (length X = 52 nm) and extended following negative staining (length X = 83 nm) or rotary shadowing (length X = 87 nm; 400-kDa core length X = 80 nm; P200 length X = 44 nm). Two or three glycosaminoglycan side chains (length X = 146 53 nm) were attached to one end of the core protein. The side chains often appeared tangled or to merge together as one. Thus, the large heparan sulfate proteoglycan from basement membrane is an asymmetrical molecule with a core protein containing globular domains and terminally attached side chains. This structure is in keeping with that previously predicted by enzymatic digestions and with the proposed orientation in basement membranes, i.e., the core protein bound in the lamina densa and the heparan sulfate side chains in the lamina lucida arranged along the surface of the basement membranes.

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“…The sections were treated with a four-layer sandwich for amplification of the signal (8) Immunogold electronmicroscopy. Immunogold electronmicroscopy was performed as described (8,23,24 Immunoblotting. Glomeruli were isolated by graded sieving, and lysates were prepared in reducing SDS sample buffer as described (8,25).…”

Section: Methodsmentioning

confidence: 99%

Proteinuria in passive Heymann nephritis is associated with lipid peroxidation and formation of adducts on type IV collagen.

Neale¹,

Ojha²,

Exner³

et al. 1994

J. Clin. Invest.

133

103

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Passive Heymann nephritis (PHN) is a model of human membranous nephropathy that is characterized by formation of granular subepithelial immune deposits in the glomerular capillary wall which results in complement activation. This is causally related to damage of the filtration barrier and subsequent proteinuria. The local accumulation of injurious reactive oxygen species (ROS) is a major effector mechanism in PHN. ROS may induce tissue damage by initiating lipid peroxidation (LPO). In turn, this leads to adduct formation between breakdown products of LPO with structural proteins, such as formation of malondialdehyde (MDA) or 4-hydroxynonenal-lysine adducts. To examine the role of LPO in the development of proteinuria we have localized MDA and 4-hydroxynoneal-lysine adducts in glomeruli of PHN rats by immunofluoresence microscopy, using specific monoclonal antibodies. By immunogold electron microscopy, MDA adducts were localized to cytoplasmic vesicles and cell membranes of glomerular epithelial cells, to the glomerular basement membrane (GBM), and also to immune deposits. Type IV collagen was specifically identified as being modified by MDA adducts, using a variety of techniques. Collagenase pretreatment of GBM extracts indicated that the NC-1 domain of type IV collagen was a site of adduct formation. When LPO was inhibited by pretreatment of PHN rats with the antioxidant probucol, proteinuria was reduced by -85%, and glomerular immunostaining for dialdehyde adducts was markedly reduced, even though the formation of immune deposits was not affected. By contrast, lowering of the serum cholesterol levels had no influence on the development of proteinuria.These findings are consistent with the premise that ROSinduced glomerular injury in PHN involves LPO and that this results not only in damage of cell membranes but in modification of type IV collagen in the GBM as well. The close temporal correlation of the occurrence of LPO with

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Immunoelectron microscopy in kidney research: Some contributions and limitations

Cited by 44 publications

References 70 publications

The Immunogold-Silver Staining Procedure in the Study of Freshly Suspended Langerhans Cells at the Transmission Electron Microscopic Level

The Immunogold-Silver Staining Procedure in the Study of Freshly Suspended Langerhans Cells at the Transmission Electron Microscopic Level

Visualization of the large heparan sulfate proteoglycan from basement membrane

Proteinuria in passive Heymann nephritis is associated with lipid peroxidation and formation of adducts on type IV collagen.

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