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PubMed1993

HMB-45 antibody demonstrates melanosome specificity by immunoelectron microscopy.

The intracellular localization of antigenic sites recognized by the monoclonal antibody HMB-45 was investigated in melanomas of the choroid and skin by postembedding immunoelectron microscopy. Antigenic sites were detected by a three-step procedure, consisting of incubating sections with the monoclonal HMB-45 antibody (protein G affinity-purified ascites from Enzo Diagnostics Inc or tissue culture supernatant from Dako Corp), followed by incubation with an affinity-purified rabbit anti-mouse IgG and finally with protein A-gold complex. Gold particles, indicative of HMB-45 immunoreactivity, were restricted to melanosomes in the malignant melanocytes. Early stages in melanosome formation (stages I through III) were most intensely stained, while late-stage melanosomes (stage IV) were only sparsely labeled or not stained at all. Melanophages adjacent to a cutaneous melanoma showed intense immunoreactivity in the cytoplasm and especially over electron-dense portions of lysosomes with the HMB-45 antibody from Enzo. In marked contrast, only very sparse labeling was detected over melanophages using a similar concentration of the HMB-45 antibody from Dako. Subsequently, when the Enzo antibody was diluted 40 times above the recommended working dilution, most of the melanophage staining disappeared, while melanocyte-specific staining was maintained. Immunolabeling of melanosomes with HMB-45 was drastically reduced or absent following section pretreatment with neuraminidase, confirming an earlier report that the HMB-45 antigen is partially composed of sialic acid. Our immunoelectron microscopic results show that HMB-45 antibody specifically stains melanosomes, rather than diffuse cytoplasmic antigen, as described by light microscopic immunohistochemical analysis, thus explaining its specificity for melanocytes. In addition, the elimination of HMB-45 immunoreactivity by neuraminidase pretreatment supports the idea that sialylation of antigen is crucial to HMB-45 binding, and suggests that the absence of staining in normal adult melanocytes, dermal nevi, and other melanocytic lesions may be a result of differential sialylation.

Antibodies, Monoclonal↗

PubMed1987

Localization of myoglobin in human muscle cells by immunoelectron microscopy.

The localization of myoglobin in human skeletal muscle cell was studied by immunoelectron microscopy. The Fab'-horseradish peroxidase (HRP) conjugate was prepared by the maleimide method recently developed by Ishikawa et al. This method gave better recovery and less polymerization of HRP than the periodate method. By the direct immunoperoxidase method using this conjugate, myoglobin was shown to be localized in the I-band region of skeletal muscle cells. The outer membrane of mitochondria and triads stained intensely, but A-bands were not stained. Myonuclei and intermyofibrillar spaces were also not stained. The localization of myoglobin in the I-band implies its function in energy generation.

Histocytochemistry↗

PubMed1976

The heterogeneity of erythrocyte antigen distribution in human normal phenotypes: an immunoelectron microscopy study.

A and A1 antigen were detected on human blood erythrocytes by immunoelectron microscopy using peroxidase-conjugated antibodies. Cells were obtained from various normal A subgroups, including rare weak A phenotypes and infant (cord blood) samples. Erythrocytes were fixed prior to incubation with specific reagents. The detection of surface antigens was carried out by an indirect method involving anti-A and anti-A1 antibodies and conjugated anti-immunoglobulin antibodies. The surface labelling was seen as a diffuse dense layer. Haemperoxidase-like activity resulted in a faint background which did not interfere at the level of ultrathin sections, with surface staining due to exogeneous peroxidase. The most significant finding was the existence, in a given sample, of several populations of cells as revealed by their antibody-binding capacity. The distribution of the various populations varied from one sample to another according to its subgroup. The progressive weakening of phenotype expression which characterizes the various subgroups from A1 to A weak was paralleled by a decreasing number of "antigen rich" cells, which were still detectable in weak phenotypes as a minor population. This study confirms that a given normal phenotype in fact represents a mixture of antigenically different populations of erythrocytes.

ABO Blood-Group System↗

PubMed1989

[An experimental investigation on application of silver-intensification technique to immunoelectron microscopy with colloidal gold probe].

The value of silver-intensification technique to immunoelectron microscopy being displayed with colloidal gold probe was evaluated through ultrastructural study on localization of cardionatrin in the atrial cardiocytes of rat. The results indicate that this technique is simple and feasible. It enables rapid screening of positively labelled sites at low magnification, and more information will be available. Further more, it offers better contrast and this technique may be of practical value.

Animals↗

PubMed1976

The cellular distribution of erythrocyte and normoblast A1 and A antigens in normal and preleukemic states. An immunoelectron microscopy study.

A1 and A alloantigens were visualized on human erythrocytes and normoblasts by immunoelectron microscopy using peroxidase-coupled antibodies. Specimens were obtained from patients with preleukemia and associated antigen weakening, and from individuals with normal antigen values. Cells were fixed by glutaraldehyde and subsequently reacted with antibodies.

ABO Blood-Group System↗

PubMed1985

Development of a sensitive protein A-gold immunoelectron microscopy method for detecting viral antigens in fluid specimens.

Protein A-colloidal gold immunoelectron microscopy (PAG IEM) has been employed to specifically detect rotavirus and enterovirus antigen in negatively stained fluid specimens. Unlike other IEM methods, PAG IEM can detect not only viral antigen associated with morphologically recognizable particles but also viral antigens of unrecognizable ultrastructure. This rapid and sensitive immunoassay was found to be applicable to virus-infected stool specimens as well as partially purified virus preparations. The sensitivity of viral antigen detection by PAG IEM was 2- to 40-fold greater than direct IEM and 200- to 1,000-fold greater than direct electron microscopy. In addition, PAG IEM appears to offer a more reliable and sensitive alternative to standard IEM for detection and quantitation of viral antibody.

Animals↗

PubMed1979

Localization of the decoding region on the 30S Escherichia coli ribosomal subunit by affinity immunoelectron microscopy.

The decoding region of the Escherichia coli ribosome has been localized by affinity immunoelectron microscopy. Valyl-tRNA1Val, derivatized at the alpha-amino end with the dinitrophenyl group, was bound to the ribosomal P site of 70S ribosomes and crosslinked specifically to 16S RNA by 310- to 325-nm irradiation. Previous work has shown that crosslink occurs between the 5' anticodon base of the tRNA and a pyrimidine in the 3' one-third of the 16S RNA. By reaction of the dinitrophenyl group with antibody, dimers of the 30S tRNA covalent complexes were prepared containing one covalently attached tRNA per 30S subunit. Electron microscopic visualization of the antibody attached to the dinitrophenyl group located the position of the 3' end of the tRNA. Several sites, with a strong preference for the large protrusion or cleft region in the upper one-third of the subunit, were found. The multiplicity of sites is likely due to the freedom of orientation of the 3' end of the tRNA which is approximately 80 A from the site of crosslinking. By extrapolating this distance from each of the antigenic sites, we concluded that the anticodon of tRNA when in the P site is probably located in the cleft region of the 30S subunit.

Dinitrophenols↗

PubMed1986

Localization of structural proteins in African swine fever virus particles by immunoelectron microscopy.

Seven African swine fever virus structural proteins were localized in the virion by immunoelectron microscopy. African swine fever virus-infected cells were incubated, before or after embedding and thin sectioning, with monoclonal antibodies specific for different structural proteins, and after labeling with protein A-gold complexes, the samples were examined in the electron microscope. Proteins p14 and p24 were found in the external region of the virion, proteins p12, p72, p17, and p37 were found in the intermediate layers, and protein p150 was found in the nucleoid and in one vertex. A monoclonal antibody that recognized protein p150 as well as p220, a virus-induced, nonstructural protein, could also bind to a component present in the nucleus of both uninfected and virus-infected cells.

African Swine Fever Virus↗

PubMed1990

[The determination of the nature of the interaction of neutral Mn2+-dependent DNAse with DNA by immunoelectron microscopy].

The interaction of neutral DNAase with native and denatured DNA was shown by immunoelectron microscopy method with the help of colloidal gold. The neutral DNAase of the rat liver nuclear chromatin is absorbed both to denatured DNA, in which the denatured regions are arranged at 5'-3' ends, and to DNA in which these regions are distributed along the whole molecule.

Animals↗

PubMed1986

Mammosomatotroph adenoma of the pituitary associated with gigantism and hyperprolactinemia. A morphological study including immunoelectron microscopy.

A 29-year old giantess with growth hormone excess and hyperprolactinemia underwent transsphenoidal surgery to remove her pituitary tumor. Electron microscopy revealed a mammosomatotroph adenoma composed of one cell type. Immunoelectron microscopy, using the immunogold technique, demonstrated predominantly growth hormone or prolactin or a varying mixture of both growth hormone and prolactin in the adenoma cells. The presence of growth hormone and prolactin was found not only in the cytoplasm of the same adenoma cells but also in the same secretory granules. In the nontumorous adenohypophysis, somatotrophs and lactotrophs showed ultrastructural signs of hyperactivity. This finding is in contrast with the presence of suppressed somatotrophs and lactotrophs seen in nontumorous portions of adult pituitaries harboring growth hormone or prolactin-secreting adenomas. Our morphological study reinforces the view that growth hormone-producing pituitary tumors, originating in childhood, are different from those of the adult gland.

Adenoma↗

PubMed1983

Glycoproteins of coated pits, cell junctions, and the entire cell surface revealed by monoclonal antibodies and immunoelectron microscopy.

Topographical descriptions of three major plasma membrane glycoproteins of murine 3T3 cells were obtained by immunoelectron microscopy with monoclonal antibodies. A glycoprotein of Mr 80,000 was distributed throughout the total cell surface. A second of Mr 90,000 was concentrated in coated pits, and a third of Mr 100,000 was localized at cell junctions.

Animals↗

PubMed1990

Studies of cicatricial pemphigoid autoantibodies using direct immunoelectron microscopy and immunoblot analysis.

We studied 11 consecutive patients with classical cicatricial pemphigoid (CP) using direct immunoelectron microscopy (IEM) and Western immunoblotting analysis. Direct IEM performed in the skin or gingival mucosa revealed in all 11 CP patients that immunoglobulins and complement deposits were usually thick and discontinuous along the dermoepidermal junction, mostly localized on the lamina densa and occasionally in the lamina lucida. By direct IEM, the ultrastructural aspect in CP differs from the pattern observed in bullous pemphigoid (BP) and from that of chronic epidermolysis bullosa acquisita (EBA). Nine CP patients were studied by Western immunoblotting and, of these nine, only two had detectable anti-basement membrane zone (BMZ) antibodies by indirect immunofluorescence on salt-split skin. By immunoblotting performed on protein extracts of heat-separated epidermis, eight out of the nine CP sera specifically reacted with two protein bands of approximately 230-240 kD and 180 kD, similar to those recognized by BP sera in co-migration experiments. By immunoblotting on skin BMZ extracts, none of these nine CP sera recognized the 290-kD major polypeptide of EBA antigen. Taken together, these results suggest that, in CP, the target-antigen, as identified on immunoblots, is similar to BP antigen, but with an abnormal expression within the dermoepidermal junction of patients, which may in part explain the scarring course of the disease.

Adult↗

PubMed1979

Immunoelectron microscopy and immunocytochemistry in pathology, with special reference to immunoglobulin-producing cells.

The advantage of immunoelectron microscopy (immuno-EM) is that it allows the simultaneous detection of surface and internal cell components. The immunoperoxidase method is often more suitable than immunoferritin. There are no major difficulties in staining surface antigens by immuno-EM, provided sufficient amounts of pure antibodies are available for coupling to peroxidase. Prior fixation of cells, with faxatives that preserve the antigenicity of surface components, avoids ligand-induced alterations of the surface components. It is believed that, unlike the surface, intracellular antigens are difficult to stain by immuno-EM because of the poor penetration of conjugates into fixed cells; thus, various technical approaches have been proposed by workers involved in tissue immuno-EM. In fact, the method that we initially devised for the surface staining of fixed cell suspensions has proved to detect specifically intracellular immunoglobulins in B cells obtained from patients with proliferative diseases. Thus, conjugates do penetrate into fixed cells, although by an unknown mechanism. On this basis, it is possible to study both surface and intracellular immunoglobulins at the EM level and to determine the precise localization synthesis.

B-Lymphocytes↗

PubMed1988

Antibody competition studies with gold-labelling immunoelectron microscopy.

Competitive studies, involving neutralizing monoclonal antibodies (NTmAbs) to outercoat proteins of Akabane virus were performed by immunoelectron microscopy. The experiments were designed to determine whether the NTmAbs were directed against the same or spatially different epitopes. Characteristics of NTmAbs in direct and indirect gold-labelling studies were determined. It was found that the protein A method gave cross-contamination of the immuno-gold complexes whereas direct conjugation of the NTmAbs to gold probes gave clean, specific and intense labelling. Analysis of dilution curves confirmed that saturation of antigenic sites did not occur and secondly determined the optimum working dilutions for the conjugated probes. The data generated in the preliminary studies enabled reliable results to be obtained from the double-labelling competitive experiment. We found that the 2 NTmAbs were directed to either the same epitope or to 2 separate but neighbouring epitopes where the binding of one NTmAb inhibited the binding of the second. The results demonstrate that if reliable data is to be obtained in double-labelling immunoelectron microscopical studies then experiments must be meticulously designed.

Antibodies, Monoclonal↗

PubMed1982

Analysis of structure-function relationships in citrate lyase isolated from Rhodopseudomonas gelatinosa as revealed by cross-linking and immunoelectron microscopy.

Citrate lyase (EC 4.1.3.6) isolated from Rhodopseudomonas gelatinosa was analyzed using cross-linking experiments and immunoelectron microscopy. Different cross-linking reagents and antibodies directed against citrate lyase and specifically against all three subunit types (L, M and S) were applied. A structure-function model is proposed for citrate lyase from R. gelatinosa: the enzyme occurs in two configurations, 'rings' and 'stars'. The ring contains two identical layers each consisting of three subunits L, with one subunit S as a polar cap sitting on each L, and three subunits M in alternating sequence (18 subunits altogether). In the star, the same 18 subunits are arranged in a different way. Whereas the subunits L are located at the periphery, the subunits M are concentrated in the center of the particle. The subunits S are positioned relative to L as in the ring; however, their location relative to the subunits M is changed. By transition from ring to star, areas on S are brought into contact with areas on M by rotation of structural units, consisting of one L, one M and one S subunit per layer, against each other, with S of one structural unit close to M of the neighbouring structural unit. This transition is assumed to work also in reversed direction. The observation of rings and stars as two distinct molecular forms is proposed to reflect the two states of citrate lyase, the ring being the form where substrate is bound by acyl exchange, and the star being the form where the substrate is consumed by cleavage, i.e. the catalyzed reaction is completed.

Cross-Linking Reagents↗

PubMed1982

A simple reliable method for producing electron dense markers of uniform size for use in immunoelectron microscopy.

A method is presented for the preparation of electron dense markers of defined size for use in immunoelectron microscopy, The electron dense markers were formed by the covalent binding of rabbit IgG and horse spleen ferritin mediated by glutaraldehyde. We demonstrated that the conjugate size, i.e. the number of ferritin grains per unit conjugate, was directly related to the time of incubation at 4 degrees C in a 1000 molar excess of glutaraldehyde. Conjugates were separated on the basis of molecular size by Sepharose 4B column chromatography. Three different sized conjugates were used to delineate pseudomonas toxin receptors on the surface of mouse LM fibroblasts; similar numbers of receptors were observed with all conjugates.

Animals↗

PubMed1984

Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold.

Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold-IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C.

Alkaline Phosphatase↗

PubMed1987

In situ localization of cartilage extracellular matrix components by immunoelectron microscopy after cryotechnical tissue processing.

Localization and distribution of proteoglycans within rat growth plate cartilage were investigated by immunoelectron microscopy. By use of a mixture of three monoclonal antibodies directed against chondroitin sulfate chains and of post-embedding staining by protein A-gold, the immunosensitivity and resolution achieved by electron microscopy within tissue processed by high-pressure freezing, freeze-substitution, and low-temperature embedding were compared with those in tissue preserved by three alternative procedures (i.e., mild chemical fixation in combination with either low-temperature embedding or conventional embedding, and high-pressure freezing and freeze-substitution followed by conventional embedding). The loss of matrix components incurred during each stage of high-pressure freezing, freeze-substitution, and low temperature embedding was also determined by measuring the loss of [35S]-proteoglycans from tissue labeled in vivo, and the results compared with previously determined estimates for tissue processed using conventional techniques. Immunosensitivity, determined as the number of gold particles per unit area, was highest in tissue processed by high-pressure freezing, freeze substitution, and low-temperature embedding. Comparable results (with a reduction of only 3-7%) were achieved within tissue preserved by mild chemical fixation followed by low-temperature embedding. In both procedures where conventional embedding was adopted, sensitivity was considerably reduced (by 51% for high-pressure freezing and freeze substitution and by 74% for mild chemical fixation). Loss of matrix components was negligible during all stages of high-pressure freezing, freeze-substitution, and low-temperature embedding. Such information, and that derived from morphological inspection of the various matrix compartments in cartilage processed by high-pressure freezing, freeze-substitution, and low-temperature embedding (J Cell Biol 98:277, 1984), together demonstrate that application of this technique results in successful immobilization of proteoglycans in situ within cartilage matrix. Although loss of proteoglycans from mildly fixed cartilage embedded under low-temperature conditions is minor, morphological examination of this tissue reveals considerable shifting of proteoglycans within matrix compartments. Hence, even though immunosensitivity may be high, resolution is poor. The beauty of the high-pressure freezing, freeze-substitution, and low-temperature embedding technique is that it combines high immunosensitivity with precise localization of matrix components at the molecular level.

Animals↗