Ultrastructural localization of Tamm—Horsfall protein in human kidney using immunogold electron microscopy

1988 ◽  
Vol 20 (3) ◽  
pp. 156-164 ◽  
Author(s):  
Robert J. Peach ◽  
William A. Day ◽  
Peter J. Ellingsen ◽  
A. Roy McGiven
Keyword(s):  
Electron Microscopy ◽  
Human Kidney ◽  
Ultrastructural Localization ◽  
Immunogold Electron Microscopy

Localization and Translocation of MMP-2 during Aggregation of Human Platelets

1998 ◽  
Vol 80 (11) ◽  
pp. 836-839 ◽  
Author(s):  
Grzegorz Sawicki ◽  
Esmond Sanders ◽  
Eduardo Salas ◽  
Mieczyslaw Wozniak ◽  
Jose Rodrigo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Flow Cytometry ◽  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Close Association ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Matrix Metalloproteinase 2 ◽  
Immunogold Electron Microscopy ◽  
Platelet Surface

SummaryWe have previously shown that human platelets express matrix metalloproteinase-2 (MMP-2) and that the release of this enzyme during platelet activation mediates the ADP- and thromboxane-independent part of aggregation. We have now used immunogold electron microscopy, flow cytometry, Western blot analysis and zymography methods to study the ultrastructural localization of MMP-2 in human washed platelets. Platelet aggregation was stimulated by collagen and the MMP-2 immunoreactivity of platelets was followed during various stages of aggregation. In resting platelets, MMP-2 was randomly distributed in the platelet cytosol without detectable association with platelet granules. Platelet aggregation caused the translocation of MMP-2 from the cytosol to the extracellular space. During the early stages of aggregation, MMP-2 remained in close association with the platelet plasma membrane. We conclude that the interactions of MMP-2 with platelet surface membranes mediate the aggregatory response induced by this enzyme.

scholarly journals Use of immunogold electron microscopy and monoclonal antibodies in the identification of nuclear substructures.

1984 ◽  
Vol 32 (7) ◽  
pp. 757-765 ◽  
Author(s):  
C V Clevenger ◽  
A L Epstein
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Ultrastructural Localization ◽  
Immunoblot Analysis ◽  
Immunogold Electron Microscopy ◽  
Supramolecular Organization ◽  
Nuclear Antigens ◽  
Interchromatin Granules ◽  
Cycle Dependent ◽  
Cytochemical Technique

A cytochemical technique for the ultrastructural localization of unique nuclear antigens is reported. Using a post-embedding indirect immunogold labeling procedure, nuclear antigens in electron-dense regions of the nucleus are localized with a minimum of nonspecific staining. Using this technique and indirect immunofluorescence, a panel of antinuclear monoclonal antibodies is shown to recognize preferentially cell cycle-dependent nuclear substructures. The antigenic domains recognized include specific regions in condensed chromatin, interchromatin granules, euchromatin, and chromosomes. The specificity of antigen recognition is demonstrated with qualitative and quantitative immunogold electron microscopy and immunoblot analysis. These results reveal the existence of previously undefined supramolecular organization within the nucleus and demonstrate the utility of the immunogold procedure when monoclonal antibodies are used.

Comparative strengths and weaknesses of peroxidase and colloidal gold in pre- and post-embedding immunolabeling techniques

1987 ◽  
Vol 45 ◽  
pp. 1002-1005
Author(s):  
D. R. Abrahamson ◽  
P. L. St.John ◽  
E. W. Perry
Keyword(s):  
Electron Microscopy ◽  
Horseradish Peroxidase ◽  
Light Microscopy ◽  
Colloidal Gold ◽  
Light Microscope ◽  
Ultrastructural Localization ◽  
The Other ◽  
Quantitative Studies ◽  
Dense Suspension ◽  
Antigen Localization

Antibodies coupled to tracers for electron microscopy have been instrumental in the ultrastructural localization of antigens within cells and tissues. Among the most popular tracers are horseradish peroxidase (HRP), an enzyme that yields an osmiophilic reaction product, and colloidal gold, an electron dense suspension of particles. Some advantages of IgG-HRP conjugates are that they are readily synthesized, relatively small, and the immunolabeling obtained in a given experiment can be evaluated in the light microscope. In contrast, colloidal gold conjugates are available in different size ranges and multiple labeling as well as quantitative studies can therefore be undertaken through particle counting. On the other hand, gold conjugates are generally larger than those of HRP but usually can not be visualized with light microscopy. Concern has been raised, however, that HRP reaction product, which is exquisitely sensitive when generated properly, may in some cases distribute to sites distant from the original binding of the conjugate and therefore result in spurious antigen localization.

Immunogold electron microscopy of surface antigens of oral bacteria

1984 ◽  
Vol 30 (8) ◽  
pp. 1008-1013 ◽  
Author(s):  
C. Mouton ◽  
L. Lamonde
Keyword(s):  
Electron Microscopy ◽  
Surface Antigens ◽  
Oral Bacteria ◽  
Immunogold Electron Microscopy ◽  
Bacterial Surface ◽  
Outer Aspect ◽  
Bacteroides Gingivalis ◽  
Cell Envelopes ◽  
Inexpensive Procedure ◽  
Monospecific Antibodies

Colloidal gold particles 3–6 nm in diameter were prepared and stabilized with the IgG fraction of polyspecific rabbit antisera produced against four different oral bacteria. The immunogold markers were used in homologous reactions to label the bacteria in a preembedding procedure for electron microscopy. An indirect immunofluorescence procedure was concurrently used to optimize the labelling conditions before observation with the electron microscope. The immunogold markers labelled fibrillar structures extending outward 50–275 nm from the Gram-positive cell envelopes and a fuzzy 5–10 nm thick capsulelike layer on the outer aspect of Bacteroides gingivalis. The immunogold method appears to be a simple, rapid, and inexpensive procedure suitable for the study of bacterial surface antigens and can be upgraded with the use of monospecific antibodies.

scholarly journals Subcellular Distribution and Relative Expression of Fibrocyte Markers in the CD/1 Mouse Cochlea Assessed by Semiquantitative Immunogold Electron Microscopy

2011 ◽  
Vol 59 (11) ◽  
pp. 984-1000 ◽  
Author(s):  
Shanthini Mahendrasingam ◽  
Catherine Bebb ◽  
Ella Shepard ◽  
David N. Furness
Keyword(s):  
Electron Microscopy ◽  
Tissue Growth ◽  
Immunogold Electron Microscopy ◽  
Spiral Ligament ◽  
Type Ii ◽  
Relative Expression ◽  
Aquaporin 1 ◽  
Cellular Transplantation ◽  
S 100 ◽  
Type V

Spiral ligament fibrocytes function in cochlear homeostasis, maintaining the endocochlear potential by participating in potassium recycling, and fibrocyte degeneration contributes to hearing loss. Their superficial location makes them amenable to replacement by cellular transplantation. Fibrocyte cultures offer one source of transplantable cells, but determining what fibrocyte types they contain and what phenotype transplanted cells may adopt is problematic. Here, we use immunogold electron microscopy to assess the relative expression of markers in native fibrocytes of the CD/1 mouse spiral ligament. Caldesmon and aquaporin 1 are expressed more in type III fibrocytes than any other type. S-100 is strongly expressed in types I, II, and V fibrocytes, and α1Na,K-ATPase is expressed strongly only in types II and V. By combining caldesmon or aquaporin 1 with S-100 and α1Na,K-ATPase, a ratiometric analysis of immunogold density distinguishes all except type II and type V fibrocytes. Other putative markers (creatine kinase BB and connective tissue growth factor) did not provide additional useful analytical attributes. By labeling serial sections or by double or triple labeling with combinations of three antibodies, this technique could be used to distinguish all except type II and type V fibrocytes in culture or after cellular transplantation into the lateral wall.

Resinless section immunogold electron microscopy of karyo-cytoskeletal frameworks of eukaryotic cells cultured in vitro. Absence of a salt-stable nuclear matrix from mouse plasmacytoma MPC-11 cells

1991 ◽  
Vol 98 (1) ◽  
pp. 107-122
Author(s):  
X. Wang ◽  
P. Traub
Keyword(s):  
Electron Microscopy ◽  
Nuclear Matrix ◽  
Protein A ◽  
Mouse Skin ◽  
Nuclear Lamina ◽  
Immunogold Electron Microscopy ◽  
Cell Structures ◽  
Critical Point Drying ◽  
Cells Cultured

The karyo-cytoskeleton of cells cultured in vitro was investigated employing resinless section immunogold electron microscopy. Cells were entrapped in low-melting agarose, sequentially extracted with various buffers and digested with nucleases to obtain karyo-cytoskeletal frameworks and reacted with specific primary and gold-conjugated secondary antibodies or gold-conjugated protein A to decorate structural elements of these frameworks. Following embedment of the gold-labeled residual cell structures in diethylene glycol distearate and their sectioning, the embedding material was removed with organic solvent and the sections were finally subjected to CO2 critical point drying. When this technique was applied to mouse skin fibroblasts (MSF), it revealed a dense and salt-stable intranuclear network of fibrogranular material. Antibodies directed against vimentin and lamin B detected a cytoplasmic meshwork of intermediate filaments (IFs) and a nuclear lamina, respectively; the latter, however, only after removal of chromatin from nuclei by nuclease digestion of DNA. Intranuclear filaments free of adhering globular material were morphologically very similar to cytoplasmic vimentin filaments. By contrast, mouse plasmacytoma MPC-11 cells lacking detectable amounts of cytoplasmic IF proteins and lamins A and C were devoid of a salt-stable internal nuclear matrix. The same holds true for MPC-11 cells that had been treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate to induce vimentin synthesis and establish a cytoplasmically extended IF network. These findings were in accordance with the biochemical behavior of Triton X-100-treated MSF and MPC-11 cells and their appearance in immunofluorescence microscopy upon extraction with high ionic strength buffer. While the chromatin was quantitatively retained in the residual cell structures derived from MSF cells, in those obtained from MPC-11 cells the nuclear lamina was disrupted and the chromatin was released from the nuclei, suggesting that MPC-11 cells lack the salt-stable nuclear scaffold to which chromatin is normally anchored.

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