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Results for “Microscopy, Immunoelectron”

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PubMed1992

Thiobacillus ferrooxidans detection using immunoelectron microscopy.

A specific, fast and very sensitive immunoelectron microscopy method was developed to morphologically and serologically distinguish different cultures of iron oxidizers. Bacteria isolated from the acidic waters of "Matahambre" and "Mina Delita" mines (Cuba) were characterized. An antiserum specific to Thiobacillus ferrooxidans did not react with other bacteria also present in the acidic waters of mine drainage. Our results suggest the occurrence of some strains of Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Leptospirillum ferrooxidans in these waters.

Antibodies, Bacterial↗

PubMedDate not supplied

Immunoelectron microscopy for chromogranin A in small cell neuroendocrine carcinoma of lung.

To see if immunoelectron microscopy can improve localization of neurosecretory granules, postembedding immunolabelling for chromogranin A was performed on 15 examples of small cell anaplastic (neuroendocrine) carcinomas primary in lung, five cases of bronchopulmonary carcinoids, and two cases of pheochromcytoma; both the carcinoids and pheochromocytomas had neurosecretory granule-rich cytoplasm by routine electron microscopy and some neurosecretory granules were identified in each of the small cell carcinomas. Immunolabeling for chromogranin A resulted in many colloidal gold particles over cytoplasmic secretory granules in both pheochromcytomas and four of the carcinoids. One carcinoid that was focally positive by immunoperoxidase staining was negative by immunoelectron microscopy. None of the 15 cases of small cell carcinoma stained for chromogranin A using immunoperoxidase techniques, but three had a small number of secretory granules weakly labeled by the anti-chromogranin A/colloidal gold complex. Immunoelectron microscopy, at least using standard glutaraldehyde fixation and epoxy resin embedding, does not increase the sensitivity of neurosecretory granule identification in small cell neuroendocrine carcinomas of the lung. Results of other studies of this neoplasm suggest that despite transcription of chromogranin genes, synthesis of the specific protein may occur to a limited extent or not at all.

Carcinoid Tumor↗

PubMed2000

Contrast-enhanced immunoelectron microscopy for Helicobacter pylori.

Since a method of contrast enhancement for immunoelectron microscopy has not been available in bacteriology, the morphological localization of proteins of Helicobacter pylori is not well known. In this report, we established a method of contrast enhancement in immunoelectron microscopy in this organism. Immunostained ultrathin sections are stained with a mixture of alcian blue and osmium tetroxide prior to staining with uranyl acetate. This method of staining provided good contrast enhancement of the bacterial cell wall and membrane without any loss of immunolabeled gold particles on the ultrathin section.

Acrylic Resins↗

PubMed1993

Antibody-induced modulation and intracellular transport of CD10 and CD19 antigens in human B-cell lines: an immunofluorescence and immunoelectron microscopy study.

Antibody-induced antigenic modulation (AIAM) of CD10 and CD19 was studied on NALM-6, RAJI, and JOK-1 cell lines using fluorescence microscopy (FM), flow cytometry (FCM), and immunoelectron microscopy (IEM). Cross-linking with monoclonal antibodies (MoAbs) induced rapid redistribution of CD10 and CD19 on the cell surface (FM) followed by internalization involving uptake through plasmalemmal pits, transfer through endosomal compartment (receptor-mediated endocytosis), and, finally, delivery to lysosomes for degradation or exocytosis and recycling (IEM). Significant quantitative differences regarding modulation and intracellular processing were shown by FCM and IEM. Thus, 35%, 30%, and 25% of CD10 compared with 80%, 60%, and 40% of CD19 were internalized in NALM-6, RAJI, and JOK-1 cells, respectively. Also, the rate of intracellular transfer as well as externalization and recycling was more pronounced in the case of CD19 than of CD10 and in the NALM-6 and RAJI cells compared with the JOK-1 cells. These differences may possibly reflect the functional significance of CD10 and CD19 as well as the stage of differentiation of the malignant B cells. Although both antigens can be useful in MoAb-targeted immunotherapy, our findings suggest that anti-CD19 MoAbs would be preferable for delivery of cytotoxic agents to malignant B cells.

Animals↗

PubMed1997

Epidermolysis bullosa acquisita diagnosed by direct immunoelectron microscopy of the conjunctiva.

OBJECTIVE: To describe for the first time the direct immunoelectron microscopic pattern of immune deposits on the conjunctival basement membrane in epidermolysis bullosa acquisita (EBA). DESIGN: Case reports. PARTICIPANTS: Two patients. INTERVENTION: Epidermolysis bullosa acquisita associated with cicatrizing conjunctivitis. MAIN OUTCOME MEASURES: Direct immunofluorescence and direct immunoelectron microscopy without freezing on conjunctival and skin biopsy specimens, indirect immunofluorescence, Western immunoblot analysis. RESULTS: Results of direct immunoelectron microscopic examination of the conjunctiva showed the presence of immune deposits in the anchoring fibril zone, just beneath the lamina densa, in both patients. This finding was the same as the direct immunoelectron microscopic pattern shown in the skin of these patients, which is known to be very specific for EBA. Direct immunofluorescence was positive in the conjunctiva of only one patient. Indirect immunofluorescence and Western immunoblot analysis failed to detect circulating autoantibodies. CONCLUSIONS: Direct immunoelectron microscopy on the conjunctiva is a useful diagnostic tool to differentiate EBA from other related autoimmune mucocutaneous blistering diseases.

Adult↗

PubMed2002

Application of immunoelectron microscopy techniques in the diagnosis of phytoplasma diseases.

An immunoelectron microscopy technique was applied to label Chrysanthemum leuchanthemum phytoplasma in infected leaf tissues of Chrysanthemum leuchanthemum L. and Catharanthus roseus L. plants. Specific monoclonal antibodies at different dilutions and secondary antimouse antibody conjugated with colloidal gold particles of different sizes were used. The monoclonal antibodies demonstrated their specificity against the antigen; immunocytological methods permitted the precise localization and identification of phytoplasmas in thin sections from infected tissues.

Antibodies, Monoclonal↗

PubMed1992

Immunoelectron microscopy of influenza A virus neuraminidase glycoprotein topography.

Using immunoelectron microscopy, the distribution of influenza A virus neuraminidase (NA) glycoproteins was examined, after performing immunoreactions to virions on the grid. With polyclonal antibody, the immunolabels of the glycoproteins were found to be homogeneously distributed, whereas with monoclonal antibody they were found to be distributed in clusters. After destruction of haemagglutinin (HA) but not of NA activity with a high concentration of trypsin, the remaining visible spikes were evenly distributed. This finding was consistent with the absence of immunolabelling with anti-HA antibody, and the homogeneous pattern of immunolabels with anti-NA polyclonal antibody, but not with the clustered labelling with the anti-NA monoclonal antibody. Thus, the immunolabelling image with anti-NA polyclonal antibody was considered to reflect the true one.

Antibodies, Monoclonal↗

PubMed1996

Examination of cutaneous macroglobulinosis by immunoelectron microscopy.

Cutaneous macroglobulinosis is a rare cutaneous manifestation of Waldenström's disease. Lesions are though to result from accumulation of macroglobulin in the dermis and are therefore called IgM storage papules. Ultrastructural findings in the previously reported cases were contradictory and the nature of the deposits was not established by electron microscopy. The purpose of this study was to analyse such deposits by the use of immunoelectron microscopy. A 60-year-old woman had multiple erythematous papules for 1 year. The histopathological changes consisted of plasmocytic infiltration of the dermis and eosinophilic deposits. The skin changes and other investigations led to a diagnosis of Waldenström's disease. Samples from normal and diseased skin were analysed by electron microscopy and by immunogold labelling with anti-IgM antibodies, after Lowicryl K4M embedding. An extracellular electron dense granular and filamentous material was observed in the mid- and upper dermis, between and within the collagen bundles. No periodicity was noted and no deposits were seen at the dermoepidermal junction. Immunoelectron microscopy showed a positive labelling located only on these deposits, in both normal-appearing skin and in lesions. In this patient, immunoelectron microscopy clearly demonstrated the presence of large amounts of IgM in the dermis, which were found in the lesions of cutaneous macroglobulinosis and in normal skin. These results suggest that the IgM storage papules result from a greater density of deposits rather than a site-specific accumulation.

Female↗

PubMed1997

The use of monoclonal antibodies with colloidal gold-labeled probes in postembedding immunoelectron microscopy.

This article focuses on procedures for the use of monoclonal antibodies (MAb) in immunoelectron microscopy (IEM) for the subcellular localization of antigens or to relate function with structure in prokaryotic and eukaryotic cells using postembedding immunoelectron microscopy techniques. The use of MAbs greatly increases the specificity and quality of information when used in combination with gold-labeled probes. Because of its specificity, the reactivity of MAb may be very sensitive to antigenic changes resulting from the process of sample preparation when performing IEM studies. Specific protocols for each particular combination of epitope/MAb must be usually specifically devised, since it is impossible to predict an experimental system that will successfully preserve structures and antigenic determinants for every combination. In this article, we discuss critical technical aspects that usually result in improved resolution when the procedure is used to identify structure in diverse prokaryotic and eukaryotic cells.

Animals↗

PubMed2001

Reelin in the extracellular matrix and dendritic spines of the cortex and hippocampus: a comparison between wild type and heterozygous reeler mice by immunoelectron microscopy.

Reelin is a glycoprotein ( approximately 400 kDa) secreted by GABAergic neurons into the extracellular matrix of the neocortex and hippocampus as well as other areas of adult rodent and nonhuman primate brains. Recent findings indicate that the heterozygote reeler mouse (haploinsufficient for the reeler gene) shares several neurochemical and behavioral abnormalities with schizophrenia and bipolar disorder with mania. These include (1) a downregulation of both reelin mRNA and the translated proteins, (2) a decrease in the number of dendritic spines in cortical and hippocampal neurons, (3) a concomitant increase in the packing density of cortical pyramidal neurons, and (4) an age-dependent decrease in prepulse inhibition of startle. Interestingly, the heterozygous reeler mouse does not exhibit the unstable gait or the neuroanatomy characteristic of the null mutant reeler mouse. Immunocytochemical studies of the expression of reelin in mice have been primarily limited to light microscopy. In this study we present new immunoelectron microscopy data that delineates the subcellular localization of reelin in the cortex and hippocampus of the wild-type mouse, and compares these results to reelin expression in the heterozygous reeler mouse. In discontinuous areas of cortical layers I and II and the inner blade area of the dentate gyrus of the wild type mouse, extracellular reelin is associated with dendrites and dendritic spine postsynaptic specializations. Similar associations have been detected in the CA1 stratum oriens and other areas of the hippocampus. In the hippocampus, reelin expression is more expansive and more widespread than in cortical layers I and II. In contrast, extracellular reelin immunoreactivity is greatly diminished in all areas examined in the heterozygous reeler mouse. However, some cell bodies of GABAergic neurons in the cortex and hippocampus demonstrate an increased accumulation of reelin in the Golgi and endoplasmic reticulum. We suggest that in the heterozygous reeler mouse a downregulation of reelin biosynthesis results in a decreased rate of secretion into the extracellular space. This inhibits dendritic spine maturation and plasticity and leads to dissociation of dendritic postsynaptic density integrity and atrophy of spines. We speculate that the haploinsufficient reeler mouse may provide a model for future studies of the role of reelin, as it may be related to psychosis vulnerability.

Animals↗

PubMed1991

Identification of coronaviruses by the use of indirect protein A-gold immunoelectron microscopy.

Concentration by airfuge and protein A-colloidal gold immunoelectron microscopy (PAG-IEM) offered a rapid and sensitive method for detection and identification of coronaviruses from various species. The method was applied to partially purified tissue culture-adapted or egg-adapted mammalian and avian coronaviruses and to clarified fecal samples from diarrheic calves and turkey poults for detection of enteric coronaviruses. Aggregates of virus coated with specific antibody were seen in virus samples mixed with homologous antiserum but not in control samples containing preexposure serum. At least a 10-50-fold enhancement of the sensitivity of direct EM for virus detection was obtained using protein A-colloidal gold complex as an electron-dense marker. The PAG-IEM method demonstrated low nonspecific background labeling and permitted detection of soluble and particle-associated antigen. Reciprocal cross-reactivity was detected among the subgroup of mammalian hemagglutinating coronaviruses, and antisera to 4 members of other subgroups only recognized their homologous virus.

Animals↗

PubMed1984

Radio frequency glow discharge and solid-phase lactoperoxidase-glucose oxidase beads as methods for etching ultra-thin plastic sections for immunoelectron microscopy.

Etching techniques to prepare ultra-thin sections for immunoelectron microscopy have incorporated a variety of reagents to expose antigenic sites. In this paper involving 2 techniques for surface etching prior to immunoelectron microscopy, radio frequency glow discharge ( RFGD ) and solid-phase lactoperoxidase-glucose oxidase beads ( Enzymobeads ) are compared to conventional peroxide etching techniques. Measuring such parameters as intensity of granule disposition and titers of antibody resulting in detectable staining. RFGD and Enzymobeads were both superior to the conventional peroxide methodology. Non-specific absorption by ferritin under the conditions utilized was not a problem with Enzymobeads or RFGD method. In addition, RFGD may be useful in situations where peroxide susceptible antigens are under study.

Adsorption↗

PubMed2004

Production of polyclonal antibodies against territrem B and detection of territrem B in the conidia of Aspergillus terreus 23-1 by immunoelectron microscopy.

Territrem B, a fungal metabolite isolated from Aspergillums terreus 23-1, is a tremorgenic mycotoxin. Immunoelectron microscopy using anti-territrem B polyclonal antibody was used to detect territrem B in the fungal body of A. terreus 23-1 at different times of culture without shaking on potato dextrose (PD) agar medium. The anti-territrem B serum was produced by immunization of rabbits with 4beta-hydroxymethyl-4beta-demethylterritrem B-sccinate bound by a linker to keyhole limpet hemocyanin. This antiserum recognized territrems and immunoelectron microscopy using this antiserum, and colloidal gold-conjugated anti-rabbit IgG antibodies showed that territrem B was localized to the fungal body of A. terreus 23-1. Territrem B was first seen in the cytoplasm of the conidia after 4 days' culture on PD agar medium. Maximal territrem B production in the conidia was seen on the 14th day of culture; however, territrem B was not formed in the hyphae at any stage of culture. These results are consistent with the previous finding that the formation of territrems is related to fungal sporulation.

Animals↗

PubMed1995

The use of Michel's transport medium for immunofluorescence and immunoelectron microscopy in autoimmune bullous diseases.

We report the use of Michel's solution, already a well established transport medium, in the combined use of direct immunofluorescence (IMF) and pre-embedding immunoelectron microscopy in 3 subepidermal bullous diseases--bullous pemphigoid (BP), epidermolysis bullosa acquisita (EBA) and dermal binding linear IgA disease (LABD). Our studies demonstrated that electron microscopy of normal skin maintained in Michel's medium for up to 28 days showed remarkable preservation of all components of the basement membrane zone, including the ultrastructure of the basal keratinocytes, dermoepidermal junction and papillary dermis. However, epidermal cell cytolysis occurred after just 48 hours. Immunoelectron microscopy using a gold probe has enabled us to localise the immunoreactants in bullous pemphigoid, epidermolysis bullosa acquisita and dermal-binding linear IgA bullous dermatoses. Our findings are comparable to and as equally reliable as those on immunoelectron microscopy of fresh skin biopsies with no loss of antigen deposition, and demonstrate an effective new use of a well established transport medium.

Epidermolysis Bullosa Acquisita↗

PubMed1997

Quantitative immunofluorescence and immunoelectron microscopy of the topoisomerase II alpha associated with nuclear matrices from wild-type and drug-resistant chinese hamster ovary cell lines.

Topo II alpha is considered an important constituent of the nuclear matrix, serving as a fastener of DNA loops to the underlying filamentous scaffolding network. To further define a mechanism of drug resistance to topo II poisons, we studied the quantity of topo II alpha associated with the nuclear matrix in drug-resistant SMR16 and parental cells in the presence and absence of VP-16. Nuclear matrices were prepared from nuclei isolated in EDTA buffer, followed by nuclease digestion with DNase II in the absence of RNase treatment and extraction with 2 M NaCl. Whole-mount spreading of residual structures permits, by means of isoform-specific antibody and colloidal-gold secondary antibodies, an estimate of the amount of topo II alpha in individual nuclear matrices. There are significant variations in topo II alpha amounts between individual nuclear matrices due to the cell cycle distribution. The parental cell line contained eight to ten times more nuclear matrix-associated topo II alpha than the resistant cell line matrices. Nuclear matrix-associated topo II alpha from wild-type and resistant cell lines correlated well with the immunofluorescent staining of the enzyme in nuclei of intact cells. The amount of DNA associated with residual nuclear structures was five times greater in the resistant cell line. This quantity of DNA was not proportional to the quantity of topo II alpha in the same matrix; in fact they were inversely related. In situ whole-mount nuclear matrix preparations were obtained from cells grown on grids and confirmed the results from labeling of isolated residual structures.

Animals↗

PubMed1992

Neuronal associations in the rat suprachiasmatic nucleus demonstrated by immunoelectron microscopy.

The synaptic associations of neurons in the suprachiasmatic nucleus (SCN) of rats were examined by single immunolabeling for somatostatin (SRIH) and arginine vasopressin (AVP), and double immunolabeling for SRIH plus AVP and vasoactive intestinal polypeptide (VIP) plus AVP. Single immunolabeling showed that SRIH neurons, which displayed some somatic and dendritic spines, formed synaptic contacts with immunonegative and positive axon terminals. AVP neurons also formed synaptic contacts with both immunonegative and positive axon terminals. The immunonegative terminals contained small, spherical clear vesicles or flattened clear vesicles. A few immunopositive AVP fibers made synapses with immunonegative somatic or dendritic spines. Double immunolabeling showed synaptic associations between SRIH axons and AVP cell bodies or dendritic processes, and between AVP axons and the somata or dendrites of SRIH neurons. These findings suggest a reciprocal relation between the two types of neurons. Synaptic contacts between AVP neurons and VIP axon terminals were also demonstrated. Previously, we found synapses between SRIH axons and VIP neurons. Thus SRIH neurons appeared to regulate AVP and VIP neurons. On the basis of these findings, two possible oscillation systems of the SCN are proposed.

Animals↗

PubMed1993

Immunoelectron microscopy identification of early proliferating cells in rat liver tissue during hyperplasia induced by lead nitrate.

Recent studies have suggested that hepatic stem cells may be involved in at least some forms of liver epithelial growth. To obtain further information on this controversial hypothesis, we treated rats with lead nitrate to induce liver growth and identified the cells undergoing early DNA synthesis by bromodeoxyuridine immunohistochemistry, using both light and electron microscopic detection methods. Eight hours after an intravenous injection of lead nitrate 100 mumol/kg, DNA synthesis was detected in a few scattered hepatocytes and in nonparenchymal cells in portal connective tissue. At the light microscopic level, identification of nonparenchymal cells was limited to bile duct epithelial cells. Other cell types were also labeled, but their identity could not be established. At the ultrastructural level, however, four types of nonparenchymal cells were identified as containing bromodeoxyuridine immunogold particles. These four types included bile duct epithelial cells, fibroblasts, macrophages and nondescript periductular cells. These periductular cells displayed certain ultrastructural features of bile duct cells but did not line a lumen or display microvilli on their apical membrane, nor did they reside within the bile duct basement membrane. Because proliferation of nonparenchymal cells in portal areas preceded that of hepatocytes, it is suggested that the former reaction reflects a direct mitogenic effect of lead nitrate and not an adaptive growth response secondary to parenchymal enlargement. However, whether DNA synthesis in periductular cells or bile duct cells reflects activation of hepatic stem cells cannot be established from the present morphological observations. If so, such a progenitor compartment must be dormant because it does not seem to play a functional role in this and other forms of adult liver epithelial growth.

Animals↗

PubMed1991

A rapid method for the preparation of yeast for immunoelectron microscopy using Lowicryl HM-20.

We describe a fixation and embedding procedure for the yeast Saccharomyces cerevisiae using Lowicryl HM-20 which is rapid, gives excellent fixation, and avoids the low temperature handling normally associated with embedding in this resin. This procedure yields superior structural preservation when compared to the commonly used rapid embedding procedure which employs Lowicryl K4M. We demonstrate that sections prepared using our rapid procedure are suitable for use in immunogold labelling experiments.

Acrylic Resins↗