scholarly journals Immune complex receptors on cell surface. I. Ultrastructural demonstration of macrophages.

1976 ◽  
Vol 24 (8) ◽  
pp. 948-955 ◽  
Author(s):  
P E McKeever ◽  
A J Garvin ◽  
S S Spicer
Keyword(s):  
Electron Microscopy ◽  
Horseradish Peroxidase ◽  
Cell Surface ◽  
Immune Complex ◽  
Immune Complexes ◽  
External Surface ◽  
Binding Capacity ◽  
Ultrastructural Localization ◽  
Soluble Complex ◽  
Receptor Sites

A method is described for ultrastructural localization of immune complex receptors on the surface of viable peritoneal exudate cells. The technique entails incubation with a soluble complex of horseradish peroxidase (HRP) and specific antibody to HRP at 4 degrees C followed by exposure to diaminobenzidine and processing for electron microscopy. The bound immune complexes were evident as focal deposits of HRP reaction product, adhering closely to the external surface of macrophages with an uninterrupted periodicity varying between 30 and 120 nm. Following incubation with an insoluble immune complex containing a higher proportion of antibody, receptor sites stained frequently, but large aggregates adhered to the cells. Rinsing cells after staining with soluble complexes partially displaced the bound immune complexes. Fixation prior to exposure to immune complexes largely eliminated the binding capacity of the immune complex receptors.

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.

Adhesion of Phytophthora palmivora zoospores: detection and ultrastructural visualization of concanavalin A-receptor sites appearing during encystment

1975 ◽  
Vol 19 (1) ◽  
pp. 11-20
Author(s):  
V.O. Sing ◽  
S. Bartnicki-Garcia
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Concanavalin A ◽  
Solid Surfaces ◽  
Trypsin Digestion ◽  
Phytophthora Palmivora ◽  
Con A ◽  
Binding Material ◽  
Ultraviolet Microscopy ◽  
Receptor Sites

The binding of concanavalin A (Con A) to the cell surface of zoospores and cysts of Phytophthora palmivora was studied by radiometry (125I-Con A), ultraviolet microscopy (fluorescein-Con A) and electron microscopy peroxidase-diaminobenzidine technique). Zoospores were found to secrete during the early stages of encystment a Con A-binding material susceptible to trypsin digestion. This glycoprotein is contained in the so-called peripheral vesicles and is probably responsible for the adhesion of the encysting zoospores to solid surfaces.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF ANTIBODY IN DIFFERENTIATING PLASMA CELLS

1968 ◽  
Vol 127 (1) ◽  
pp. 109-118 ◽  
Author(s):  
Elizabeth H. Leduc ◽  
Stratis Avrameas ◽  
Michel Bouteille
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Horseradish Peroxidase ◽  
Peroxidase Activity ◽  
Plasma Cells ◽  
Ultrastructural Localization ◽  
Fixed Tissue ◽  
Plasmacytic Differentiation ◽  
Perinuclear Space ◽  
Large Spherical

Antibody was localized by electron microscopy within differentiating and mature plasma cells of the spleens of hyperimmunized rabbits. Horseradish peroxidase was used as antigen. Intracellular antibody to peroxidase was revealed in glutaraldehyde-fixed tissue by coupling it with its antigen and then revealing the sites of peroxidase activity cytochemically. Antibody first appears in the perinuclear space of hemocytoblasts where it persists through differentiation into immature plasma cells, but it disappears from this site in mature plasma cells. Concomitant with the development of the ergastoplasm, antibody accumulates in many but not all of its cisternae. Antibody is present in the lamellar portion of the Golgi apparatus in all phases of plasmacytic differentiation. Mature plasma cells exhibit two types of antibody distribution, a concentration into large spherical intracisternal granules or an overflowing into all parts of the cytoplasm.

scholarly journals Insulin-induced receptor loss in the cultured human lymphocyte: quantitative morphological perturbations in the cell and plasma membrane

1979 ◽  
Vol 39 (1) ◽  
pp. 77-88
Author(s):  
P. Gordon ◽  
J.L. Carpentier ◽  
E. Van Obberghen ◽  
P. Barazzone ◽  
J. Roth ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Human Lymphocyte ◽  
Human Lymphocytes ◽  
Experimental Conditions ◽  
Peroxidase Uptake

When cultured human lymphocytes (IM-9) are exposed to 10(−6) M procine insulin for 6 h, washed, and incubated with 125I-insulin, the ability of the cell to bind the labelled hormone is reduced by a mean of 78%. Under these experimental conditions that induce insulin-receptor loss in this cell there is a mean 95% increase in microinvaginations in the plasma membrane revealed by electron microscopy on freez-fractured replicas of the cell. At the same time, horseradish peroxidase uptake, a marker of endocytosis, is increased in the cells incubated with insulin. Coupled with our recent EM autoradiographic evidence that labelled insulin is acutely internalized by this cell, these studies are consistent with the possibility that endocytosis represents a mechanism by which receptor is removed from the cell surface.

Scanning and Transmission Microscopy of Nematocyst Batteries in Epitheliomuscular Cells of Hydra

1971 ◽  
Vol 29 ◽  
pp. 410-411 ◽  
Author(s):  
Jane A. Westfall ◽  
S. Yamataka ◽  
Paul D. Enos
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Osmium Tetroxide ◽  
External Surface ◽  
Three Dimensional ◽  
Surface Structures ◽  
Transmission Microscopy ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Electron

Scanning electron microscopy (SEM) provides three dimensional details of external surface structures and supplements ultrastructural information provided by transmission electron microscopy (TEM). Animals composed of watery jellylike tissues such as hydras and other coelenterates have not been considered suitable for SEM studies because of the difficulty in preserving such organisms in a normal state. This study demonstrates 1) the successful use of SEM on such tissue, and 2) the unique arrangement of batteries of nematocysts within large epitheliomuscular cells on tentacles of Hydra littoralis.Whole specimens of Hydra were prepared for SEM (Figs. 1 and 2) by the fix, freeze-dry, coat technique of Small and Màrszalek. The specimens were fixed in osmium tetroxide and mercuric chloride, freeze-dried in vacuo on a prechilled 1 Kg brass block, and coated with gold-palladium. Tissues for TEM (Figs. 3 and 4) were fixed in glutaraldehyde followed by osmium tetroxide. Scanning micrographs were taken on a Cambridge Stereoscan Mark II A microscope at 10 KV and transmission micrographs were taken on an RCA EMU 3G microscope (Fig. 3) or on a Hitachi HU 11B microscope (Fig. 4).

A Sensitive On-Grid Immunoelectron Microscopic Procedure for Diagnosis of Rotavirus Infection

1980 ◽  
Vol 38 ◽  
pp. 506-507
Author(s):  
M. F. Miller ◽  
A. R. Rubenstein
Keyword(s):  
Electron Microscopy ◽  
Specific Antibody ◽  
Immune Complexes ◽  
Acute Gastroenteritis ◽  
Plant Viruses ◽  
Aggregation Process ◽  
Microscopic Technique ◽  
Fecal Material ◽  
Present Communication ◽  
Number Of Particles

Studies of rotavirus particles in humans, monkeys and various non-primates with acute gastroenteritis have involved detection of virus in fecal material by electron microscopy. The EM techniques most commonly employed have been the conventional negative staining (Fig. 1) and immune aggregation (Fig. 2) procedures. Both methods are somewhat insensitive and can most reliably be applied to samples containing large quantities of virus either naturaLly or as a result of concentration by ultracentrifugation. The formation of immune complexes by specific antibody in the immune aggregation procedures confirms the rotavirus diagnosis, but the number of particles per given microscope field is effectively reduced by the aggregation process. In the present communication, we describe use of an on-grid immunoelectron microscopic technique in which rotavirus particles are mounted onto microscope grids that were pre-coated with specific antibody. The technique is a modification of a method originalLy introduced by Derrick (1) for studies of plant viruses.

Localization of Immune Complexes in the Basement Membrane of the Ductuli Efferentes of the Rhesus Monkey

1980 ◽  
Vol 38 ◽  
pp. 456-457
Author(s):  
D. Marsh
Keyword(s):  
Basement Membrane ◽  
Fluorescence Microscopy ◽  
Rhesus Monkey ◽  
Immune Complex ◽  
Immune Complexes ◽  
Vas Deferens ◽  
Frozen Sections ◽  
Efferent Ducts ◽  
Complex Deposition ◽  
Sperm Antibodies

As a result of vasectomy, spermatozoa are confined to the epididymis and vas deferens, where they degenerate, releasing antigens that enter the circulation or are engulfed by macrophages. Multiple antigens of the sperm can elicit production of autoantibodies; circulating anti-sperm antibodies are found in a large percentage of vasectomized men, indicating the immunogenicity of the sperm. The increased prevalence of macrophages in the liomen of the rhesus monkey testicular efferent ducts after vasectomy led to further study of this region. Frozen sections were used for evaluation of immunopathological status by fluorescence microscopy with fluorescein-conjugated antibody. Subsequent granular deposits of immune complexes were revealed by positive immunofluorescence staining for complement. The immune complex deposition in the basement membrane surrounding the efferent ducts implies that this region is involved in antigen leakage (Fig. 1).

The Mesothelial Cell as a Non-Thrombogenic Surface

1984 ◽  
Vol 52 (02) ◽  
pp. 102-104 ◽  
Author(s):  
L J Nicholson ◽  
J M F Clarke ◽  
R M Pittilo ◽  
S J Machin ◽  
N Woolf
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Blood Flow ◽  
Cell Surface ◽  
Mesothelial Cell ◽  
Mesothelial Cells ◽  
Plastic Film ◽  
In Vitro System ◽  
Scanning Electron

SummaryA technique for harvesting mesothelial cells is described. This entails collagenase digestion of omentum after which the cells can be cultured. The technique has been developed using the rat, but has also been successfully applied to human tissue. Cultured rat mesothelial cells obtained in this way have been examined by scanning electron microscopy. Rat mesothelial cells grown on plastic film have been exposed to blood in an in vitro system using a Baumgartner chamber and have been demonstrated to support blood flow. No adhering platelets were observed on the mesothelial cell surface. Fibroblasts similarily exposed to blood as a control were washed off the plastic.

Competitive enzyme-liked immunoassay with use of soluble enzyme/antibody immune complexes for labeling. I. Measurement of human choriogonadotropin.

1976 ◽  
Vol 22 (8) ◽  
pp. 1372-1377 ◽  
Author(s):  
D E Yorde ◽  
E A Sasse ◽  
T Y Wang ◽  
R O Hussa ◽  
J C Garancis
Keyword(s):  
Horseradish Peroxidase ◽  
Peroxidase Activity ◽  
Quantitative Measurement ◽  
Original Sample ◽  
Soluble Enzyme ◽  
Soluble Complex ◽  
Human Choriogonadotropin ◽  
Preliminary Study ◽  
Free Antigen ◽  
Serum And Urine

Abstract We described the principle of a new enzyme-immunoassay, competitive enzyme-liked immunoassay (CELIA), for quantitative measurement of soluble antigens and haptens. In the assay, binding of antibody to antigen-immunosorbent is competitively inhibited by the free antigen to be measured. The amount of first antibody bound to the immunosorbent is measured by an enzymatic technique in which a heterologous bridging antibody and a soluble antibody/enzyme immune complex are applied in sequence. The soluble complex we used was rabbit antiperoxidase/horseradish peroxidase. Peroxidase activity is inversely proportional to the concentration in the original sample of the substance to be assayed. The enzyme-linked reagents are potentially widely applicable to any substance to be measured. To demonstrate the feasibility of CELIA, we report a preliminary study of its application to the measurement of human chloriogonadotropin in serum and urine. The assay described for this hormone has a working range of 1 to 50 int. units per milliliter of sample. The technique obviates the disadvantages associated with measurement and handling of radioisotopes in radioimmunoassays and the only major instrumentation required is a centrifuge and a conventional spectrophotometer.

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