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Results for “Microscopy, Immunoelectron”

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PubMed1990

Propylthiouracyl-induced hypothyroidism results in reversible transdifferentiation of somatotrophs into thyroidectomy cells. A morphologic study of the rat pituitary including immunoelectron microscopy.

Two-month-old female Fischer-344 rats were rendered hypothyroid by ingestion of propyl-thiouracyl (PTU) (0.1% in drinking water) and sacrificed 3, 7, 14, and 28 days after the start of PTU administration as well as 3, 7 and 14 days after interruption of a 14-day PTU treatment. Controls received no PTU. The pituitaries were studied by histology, immunohistochemistry, electron microscopy, and immunoelectron microscopy, using the immunogold double-labeling technique. In the course of hypothyroidism, pituitary thyrotrophs had undergone the well-known thyroidectomy change. In addition, a contingent of growth hormone (GH) cells lost their large secretory granules, enlarged, displayed progressive dilation of rough endoplasmic reticulum, thereby transforming into thyroidectomy cells. These bihormonal thyrosomatotrophs contained gH in their secretory granules and thyrotropin in the dilated rough endoplasmic reticulum as documented by the immunogold double-labeling method for GH and thyrotropin. After discontinuation of PTU treatment, a rapid increase in size, number and GH labeling of secretory granules and simultaneous involution of distended rough endoplasmic reticulum with reduction of thyrotropin labeling took place in thyrosomatotrophs. A practically complete restitution of normal pituitary structure was seen in 2 weeks. Results implicate that, contrary to previously accepted concepts, adenohypophysial cells may not be irreversibly committed to one morphologically recognizable cell line.

Animals↗

PubMed1994

Altered collagen expression in human dentin: increased reactivity of type III and presence of type VI in dentinogenesis imperfecta, as revealed by immunoelectron microscopy.

We used transmission immunoelectron microscopy and polyclonal antibodies to study the reactivities of Types III and VI collagen in dentin of normal human permanent and primary teeth and in primary teeth from five patients with dentinogenesis imperfecta (DI) associated with osteogenesis imperfecta and occurring as a single trait. In the normal permanent tooth, reactivity of Type III collagen was occasional and, where present, peritubular. Staining of normal primary teeth was less occasional but still rare, whereas the abnormal dentin stained more uniformly. Atypical, non-striated fibrillar structures that also showed Type III collagen reactivity were observed in dentin of two of the three patients with DI as a single trait. Later, these two patients proved to be first cousins. Unlike antibodies to the N-terminal pro-peptide of Type I pro-collagen, antibodies to the C-terminal telopeptide of Type I collagen, used for comparison stained the affected dentin homogeneously. Reactivity of Type VI collagen, not detected in normal teeth, was seen in the dentin of all abnormal teeth, in association with non-fibrillar delicate material. This study also shows that although readily detectable in dentin affected by DI, Type III collagen is a minor constituent of normal human dentin matrix.

Collagen↗

PubMedDate not supplied

Large block embedding and "pop-off" technique for immunoelectron microscopy.

A technique for immunoelectron microscopy is described whereby tissue is embedded in large (6 x 12 mm) plastic blocks and reembedded ("popped off") directly from semithin (3-microns) sections for thin sectioning. This technique is particularly useful in locating relatively sparse cells or tissue structures. Three acrylic plastics were evaluated: Lowicryl K4M, LR Gold, and LR White. Results indicated excellent ultrastructural morphologic preservation and retention of antigenicity with the first two plastics both comparable to conventional methods of processing. Lowicryl and LR Gold were successfully adapted to the large block pop-off technique. Technical problems were encountered with LR White.

Acrylic Resins↗

PubMed1990

Localization of an intermediate chain of outer arm dynein by immunoelectron microscopy.

We have used immunoelectron microscopy to determine the location of an intermediate chain in the isolated outer arm dynein from Chlamydomonas flagella. When the purified alpha beta dimer of the outer arm was incubated with antibodies recognizing two distinct epitopes on its 69-kDa intermediate chain and then negatively stained and examined by electron microscopy, both antibodies appeared to have bound to the base of the Y-shaped stem that connects the two heads of the particle. These results indicate that this intermediate chain is located at the base of the stem. Inasmuch as this polypeptide is tightly associated with the 78-kDa intermediate chain and several light chains in an intermediate chain-light chain complex, it is likely that this entire assemblage is located at the base of the particle. Thus, these polypeptides are in a potentially important position with regard to the ATP-insensitive (structural end) binding of dynein to microtubules and to dynein-dynein interactions within the axoneme.

Antibodies, Monoclonal↗

PubMed1996

Identification of Natural Killer Cells by Immunoelectron Microscopy

We employed immunoelectron microscopic techniques to localize natural killer cells (NK cells) in human lymph node and tonsil. These tissues were embedded in Lowicryl K4M. Thin sections were first reacted with anti-Leu-7 followed by anti-UCHL. Colloidal gold particles of different sizes were used as a label. NK cells were localized primarily in paracortical T-cell regions. The cells typed with these antisera include both large granular and agranular lymphocytes. No other cell types expressed the NK phenotype. These results illustrate the versatility of immunoelectron microscopy to solve problems beyond the resolution of the light microscope.

Journal Article↗

PubMed1981

Localization of the puromycin binding site on the large ribosomal subunit of Escherichia coli by immunoelectron microscopy.

By using immunoelectron microscopy, we have localized the binding site on 50S Escherichia coli ribosomal subunits for puromycin, an antibiotic that interacts with the ribosomal peptidyltransferase center. This was achieved by affinity-labeling 50S subunits with N-bromoacetyl puromycin and treating the labeled subunits with an antibody specific for the N6,N6-dimethyladenosine moiety of puromycin. The position of the puromycin binding site was then revealed by localizing the attachment sites of the IgG molecules on the surfaces of the 50S subunits under the electron microscope: it was located at the interface between the subunits, on and around the wider lateral protuberance of the 50S subunit. This localizes directly the peptidyl transferase center on the surface of the large ribosomal subunit.

Binding Sites↗

PubMed1988

Selective induction of peroxisomal enzymes by the hypolipidemic drug bezafibrate. Detection of modulations by automatic image analysis in conjunction with immunoelectron microscopy and immunoblotting.

Quantitative immunoelectron microscopy in conjunction with quantitative analysis of immunoblots have been used to study the effects of bezafibrate (BF), a peroxisome-proliferating hypolipidemic drug, upon six different enzyme proteins in rat liver peroxisomes (Po). Antibodies against following peroxisomal enzymes: catalase, urate oxidase, alpha-hydroxy acid oxidase, acyl-CoA oxidase, bifunctional enzyme (hydratase-dehydrogenase) and thiolase, were raised in rabbits, and their monospecificities were confirmed by immunoblotting. Female Sprague-Dawley rats were treated for 7 days with 250 mg/kg/day bezafibrate and liver sections were incubated with the appropriate antibodies followed by the protein A-gold complex. The labeling density for each enzyme was estimated by automatic image analysis. In parallel experiments immunoblots prepared from highly purified peroxisome fractions of normal and BF-treated rats were incubated with the same antibodies. The antigens were visualized by an improved protein A-gold method including an anti-protein A step and silver amplification. The immunoblots were also quantitated by an image analyzer. The results revealed a selective induction of beta-oxidation enzymes by bezafibrate with thiolase showing the most increase followed by bifunctional protein and acyl-CoA oxidase. The labeling density for catalase and alpha-hydroxy acid oxidase was reduced, confirming fully the quantitative analysis of immunoblots which in addition revealed reduction of uricase. These observations demonstrate that hypolipidemic drugs induce selectively the beta-oxidation enzymes while other peroxisomal enzymes are reduced. The quantitative immunoelectron microscopy with automatic image analysis provides a versatile, highly sensitive and efficient method for rapid detection of modulations of individual proteins in peroxisomes.

Acetyl-CoA C-Acetyltransferase↗

PubMed2000

Melanocytes in nevi and melanomas synthesize basement membrane and basement membrane-like material. An immunohistochemical and electron microscopic study including immunoelectron microscopy.

Light microscopic studies have shown that nevus cell nests and melanoma nests are surrounded by basement membrane (BM) material containing type IV collagen and laminin. This study confirms this by electron microscopy and relates it to proteins which interact with the basement membrane. Nevi except for dysplastic and Spitz nevi, malignant melanomas, and melanoma metastases were studied by immunohistopathology, routine electron microscopy (EM), and immunoelectron microscopy. The lesions were incubated with monoclonal antibody (moAb) against type IV collagen, laminin, and the integrin alpha6 and studied by light microscopy. In addition, melanomas were studied by immuno-EM after incubation with a moAb against matrix metalloproteinase-2 (MMP-2). Nevus cell nests and melanoma nests are surrounded by BM material containing type IV collagen and laminin by immuno-EM. The BM material various in thickness and is amorphous. Type IV collagen, laminin, and MMP-2 are synthesized by melanoma cells as well as adjacent fibroblasts. Destruction or loss of the BM is not mandatory for melanoma invasion or even metastasis. Possibly the BM material is a protective wall for melanoma cells. Interactions between melanocytes and the extracellular matrix of which the BM is a part, can be traced back to the migration of melanocytes from the neural crest.

Antibodies, Monoclonal↗

PubMed1997

Displacement of gold marker in immunoelectron microscopy of human respiratory cilia.

Preembedding immunogold electron microscopy was performed to evaluate the position of outer arm dynein heavy chains in normal human respiratory cilia. Anti-dynein antibody (AD2), which is specific for sea urchin sperm flagellar dynein heavy chains, was used as primary antibody. Direct cross-sections of cilia were selected, and the distance between the center of a cilium and the center of a colloidal gold particle attached to the cilium (X) was measured. The distance between the center of a cilium and the farthest edge of an outer dynein arm of the cilium was measured by ordinary electron microscopy (Yo) and by immunoelectron microscopy (Yi). X was significantly longer than Yo and Yi. If it is assumed that the structure of respiratory cilia is dense and that antibodies are located at the outer side of the actual position of the heavy chains, then the average distance difference of approximately 90-120 A may represent the length of two conjugated antibodies. This length should be kept in mind when performing immunoelectron microscopy. The data suggest that AD2 recognizes the outer arm dynein heavy chains of normal human respiratory cilia.

Adult↗

PubMed1991

Processing of proopiomelanocortin (POMC) approached by immunoelectron microscopy pre-embedding method.

Immunoelectron microscopy pre-embedding method was applied to study the processing of proopiomelanocortin (POMC). Premature pituitary cells and cancer cells showed POMC derived peptides in perinuclear spaces (PNS), rough endoplasmic reticula (RER) and in a few secretory granules (SG). These cells demonstrated immature processing of POMC and suggested constitutive pathway. Cultured mouse fibroblasts transfected with human POMC gene was a good model for this system. Mature pituitary cells and pituitary adenomas showed POMC derived peptides predominantly in SG which correlated with authentic processing of POMC. Cultured mouse AtT 20 cells transfected with human POMC gene showed similar secretory pathway and processing. These results emphasized that SG is mandatory for proper processing of POMC. SG is probably essential for the regulated system of secretion.

Adrenocorticotropic Hormone↗

PubMed1999

Update on immunoelectron microscopy in diagnostic dermatopathology and cutaneous biology.

Immunoelectron microscopy for diagnostic dermatopathology is hardly ever necessary. Immunoelectron microscopy, however, is very helpful in the work-up of interesting cases, for the study of genetic and autoimmune bullous diseases and for basic science studies, particularly in conjunction with molecular biology techniques. It is also very helpful for investigators requiring double labeling.

Female↗

PubMed1994

Immunoelectron microscopy of different forms of glomerulonephritis in routine biopsy material.

Renal biopsies were investigated of patients with IgA or membraneous glomerulonephritis or with systemic lupus erythematosus by light microscopy, electron microscopy, light microscopic immunohistology and by immunoelectron microscopy using the post-embedding technique applied to LR-White embedded tissue. Aim of the study was to explore whether immunoelectron microscopy is reproduced on routine biopsy material and in accordance with light microscopic immunohistological findings. The study shows that immunoelectron microscopy can be applied to routine biopsy material and gives reproducible results. The applied method proved to be reliable, and, hence, routine biopsy material may be used for further studies concerning subcellular mechanisms in immunocomplex deposition and removal.

Biopsy, Needle↗

PubMed1992

Immunoelectron microscopy of enzymes, multienzyme complexes, and selected other oligomeric proteins.

The collective term "immunoelectron microscopy" subsumes a number of techniques in which the biological material is decorated with specific antibodies, prior to being visualized in the electron microscope. In this article, we have reviewed literature on immunoelectron microscopy that focusses on the analysis of the molecular architecture of proteins, in particular of enzymes and of multienzyme complexes. Molecular immunoelectron microscopy has been remarkably successful with multi-subunit enzymes of complex quaternary structures, and in many cases the data have been the basis for the eventual development of detailed three-dimensional molecular models. The elucidation of subunit composition and juxtaposition of a given enzyme, an important accomplishment in itself, has in turn stimulated and guided discussions on the catalytic mechanism; illustrative examples include F1 ATPase and citrate lyase, among others. Here we have chosen a variety of enzymes, multienzyme complexes, and non-enzymatic proteins to demonstrate the versatility of immunoelectron microscopy, to illustrate methodological prerequisites and limitations, and to discuss significance and implications of individual immunoelectron microscopy studies.

Animals↗

PubMed1991

Immunoelectron microscopy shows an atypical pattern and a quantitative shift of collagens type I, III and VI in oral Kaposi's sarcoma of AIDS.

The localization of collagen types I, III and VI in normal human alveolar and palatal mucosa and in oral Kaposi's sarcoma (KS) was studied by light microscopy and cryo-immunoelectron microscopy. Normal oral mucosa revealed two different types of organization. The upper connective tissue stroma contained a loose reticular network mainly composed of collagen types III and VI, while collagen type I immunostaining predominated in the deeper stroma. Ultrastructurally, in the KS tumour stroma, a loose pattern of individual thin collagen fibrils was noted. These often fanned out at their ends showing a filamentous substructure. The fibrils consisted predominantly of collagen type I similar to individual fibrils of normal oral mucosa. However, there was a marked loss of thick fibre bundles of collagen types I and III in KS compared with normal oral mucosa, whereas collagen type VI was markedly increased and found preferentially in clusters and strands around cross-striated fibrils that often spanned the distance between single collagen fibres. The abundance of collagen type VI in a pattern similar to early stages of wound healing suggests that the KS stroma resembles an early organizational stage of the interstitial and vascular extracellular matrix subject to a high rate of collagen turnover. This character of the KS stroma appears to result from a continuous auto-and paracrine stimulation of cell growth and collagen synthesis and provides an excellent model to study the structural arrangement of collagen type VI in relation to the fibrillar collagen types I and III.

Acquired Immunodeficiency Syndrome↗

PubMed2001

Effects of mutant Vpr/Vpx on HIV-2 assembly demonstrated by immunoelectron microscopy.

The virion-associated accessory proteins Vpr and Vpx of human immunodeficiency virus type 2 (HIV-2) are required for efficient viral replication. Vpr could be important for Vpx assembly. To investigate the interaction of Vpr and Vpx with respect to the effects of reverse transcriptase (RT) activity, viral particle information and Vpx expression site directed mutagenesis was carried out to construct Vpr, Vpx and double Vpr/Vpx HIV-2 mutants. These mutants were used for infection of peripheral blood mononuclear cells (PBM), human acute lymphoblastomic leukaemia cells (CEM-CM3) and HeLa CD4+ cells. Visualization of Vpx expression was carried out using FITC and gold labelling by means of laser scanning confocal microscopy and semi quantitative immunoelectron microscopy. Intracellular and extracellular localizations of Vpx were determined by means of fine structural analysis. Up to 80-90% reduction in the RT activity, total number of viral particles, and average Vpx expression was observed after infection of target cells with the Vpr mutant strains. In addition, intracellular Vpx expression was reduced to 51.2% with the Vpr mutant. Only 0.02% Vpx expression was detected after mutation at amino acid 62. These results provide evidence that Vpr or Vpr/Vpx mutants reduce RT activity and interfere with the expression of Vpx in HIV-2 particles during viral assembly. Vpr is efficient for Vpx corporation during viral assembly.

CD4 Antigens↗

PubMed1993

The globular head domain of titin extends into the center of the sarcomeric M band. cDNA cloning, epitope mapping and immunoelectron microscopy of two titin-associated proteins.

Immunoelectron microscopical results have shown that the Z and M bands of the sarcomere are interconnected by the long titin molecules. Here we have characterized by monoclonal antibodies, cDNA cloning and immunoelectron microscopy the two titin-associated proteins (190 and 165 kDa proteins), which seem responsible for the formation of a head structure on one end of the 0.9 micron long titin string. The human 165 kDa (1465 residues) and 190 kDa (1451 residues) proteins have unique N-terminal domains some 110 residues in length. Both proteins show 12 repeat domains with strong homology to either fibronectin type III (motif I) or immunoglobulin C2 (motif II) domains, which are arranged in the order II-II-I-I-I-I-I-II-II-II-II-II. Over these repeat domains the two proteins share 50% sequence identity (70% similarity). Epitopes situated in the C-terminal 138 or in the preceding 206 residues of the 165 kDa protein locate in immunoelectron microscopy to stripes situated 18 or 15 nm from the center of the M band. An epitope situated 277 to 129 residues prior to the C-terminus of the 190 kDa protein (i.e. repeats 10 and 11) locates to the center of the M band. Thus the head structure of the titin molecule extends into the center of the M band. Microsequence data on peptides from the titin-associated bovine 165 kDa protein and from conventionally purified bovine M-protein argue together with the reactivity of the antibodies that 165 kDa protein and M-protein are identical. The integrating structure of the sarcomere, which is based on titin and its side-on (C-protein and 86 kDa protein) or end-on (190 kDa protein and 165 kDa protein) associated proteins arises from muscle-specific members of the superfamily of immunoglobulin-like proteins.

Amino Acid Sequence↗

PubMed1993

Use of immunoelectron microscopy to demonstrate Francisella tularensis.

Three immunoelectron microscopy (IEM) methods were employed to show laboratory-cultivated Francisella tularensis. By the IEM assays, F. tularensis was distinguished from four antigenically distinct gram-negative bacteria. IEM should be a valuable tool for confirming presumptive isolates of F. tularensis and may potentially be useful for demonstrating other medically important bacteria.

Antigens, Fungal↗

PubMed1995

Resin embedding for quantitative immunoelectron microscopy. A comparative computerized image analysis.

Quantitative immunoelectron microscopy (QIEM) is dependent on the reliability of preparative techniques for both efficient immunolabeling and consistent quantitative results among series of immunostained sections. The present study compared Lowicryl K4M and Epon embedding after identical fixation and dehydration of rat somatotrophic secretory granules. Labeling intensity, diameter, roundness, uptake of uranyl acetate, and gray value were measured with computer assisted image analysis. Lowicryl-embedded granules showed the highest labeling densities after conventional fixation and Progressively Lowering Temperature (PLT) dehydration, but values were not consistent in a series of immunostained sections. A lower but more uniform level of immunostaining was seen in Epon-embedded sections when tissue was cryofixed and physically dehydrated. Gray value measurements from micrographs from both embedding media confirmed the better contrast of Epon sections and the different reliefs of the granule surfaces. This study emphasizes the importance of complete comparisons of preparative techniques for QIEM for reliability and reproducibility.

Animals↗