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PubMed1987

Immunoelectron microscopy for growth hormone and prolactin in pituitary adenomas.

Growth hormone (GH)- and prolactin (Prl)-producing pituitary adenomas were studied with immunoelectron microscopy using protein A-gold complex, and were compared with the normal pituitary gland. GH-producing cells were readily identifiable by numerous, uniformly dense, round secretory granules in both adenomas and normal pituitary gland. In contrast, Prl secretory granules in normal pituitary gland were much larger in size than the scarce, smaller, secretory granules of Prl-producing adenomas. Thus immunoelectron microscopic identification of Prl is more valuable for prolactinoma. With more specific antigens available as tumor markers, immunoelectron microscopy appears to be a powerful tool for tumor diagnosis.

Adenoma↗

PubMed2001

Comparison in localization between cystatin C and cathepsin K in osteoclasts and other cells in mouse tibia epiphysis by immunolight and immunoelectron microscopy.

We compared the distribution of a cysteine proteinase inhibitor, cystatin C, with that of cathepsin K in osteoclasts of the mouse tibia by immunolight and immunoelectron microscopy. Light microscopically, strong immunoreactivity for cystatin C was found extracellularly along the resorption lacuna and intracellularly in the organelles of osteoclasts. In serial sections, various patterns of cystatin C and cathepsin K localization were seen, specifically: (1) some resorption lacuna were positive for both cystatin C and cathepsin K; (2) others were positive for either cystatin C or cathepsin K, but not both; and (3) some lacuna were negative for both. In osteoclasts, the localization of cystatin C was similar to that of cathepsin K. Furthermore, cystatin C immunoreactivity was detected in preosteoclasts and osteoblasts, whereas cathepsin K was seen only in preosteoclasts. Electron microscopically, cystatin C immunoreactive products were found in the rough endoplasmic reticulum (ER), Golgi apparatus, vesicles, granules, and vacuoles of osteoclasts. These cystatin C-positive vesicles had fused or were in the process of fusion with the ampullar vacuoles (extracellular spaces) containing cystatin C-positive, fragmented, fibril-like structures. The extracellular cystatin C was deposited on and between the cytoplasmic processes of ruffled borders, and on and between type I collagen fibrils. In the basolateral region of osteoclasts, cystatin C-positive vesicles and granules also fused with vacuoles that contained cystatin C-positive or negative fibril-like structures. These results indicate that osteoclasts not only synthesize and secrete cathepsin K from the ruffled border into the bone resorption lacunae, but also a cysteine proteinase inhibitor, cystatin C. Therefore, it is suggested that cystatin C regulates the degradation of bone matrix by cathepsin K, both extracellularly and intracellularly.

Animals↗

PubMed1991

Antigenic differences between Coxiella burnetii cells revealed by postembedding immunoelectron microscopy and immunoblotting.

The aim of this study was to investigate the antigenic structures of the morphologically distinct cells of the Coxiella burnetii developmental cycle. Postembedding immunoelectron microscopy with polyclonal antibodies produced in rabbits to (i) phase I cells, (ii) a chloroform-methanol residue fraction of cells, (iii) the cell walls (CW) of large and small cells and small dense cells (SDC), and (iv) the peptidoglycan-protein complexes of small cells and SDC labelled the continuum of morphologically distinct cells. But these antibodies did not distinguish between the antigenic structures of the various cells. Monoclonal antibodies to the phase I lipopolysaccharide labelled the CW of a majority of the smaller cells, but there was diminished reactivity to the larger cells. Although monoclonal antibodies to a 29.5-kDa outer membrane protein labelled the CW of the large mother cells, the large cells, and the small cells, a minority of the SDC with compact CW were not labelled. The endogenous spore within the mother cell was not labelled by the polyclonal or monoclonal antibodies to cellular components. A selected population of SDC was prepared by osmotic lysis of large cells, differential centrifugation, Renografin step-gradient fractionation, and breakage of the small cells in a French press at 20,000 lb/in2. The pressure-resistant SDC collected as fraction CL did not contain the 29.5-kDa protein, as evidenced by the lack of (i) Coomassie brilliant blue staining of protein in the 29.5-kDa region of sodium dodecyl sulfate-polyacrylamide gels and (ii) reactivity of the 29.5-kDa protein antigenic epitopes in immunoblotting with monoclonal antibodies to the protein. In contrast, CW of the pressure-sensitive small cells contained the 29.5-kDa protein. Therefore, the observed ultrastructural differences between large and small cells and SDC reflect differences in sensitivity to breakage by pressure treatment and in cell-associated antigens. Although the process of differentiation in C. burnetii remains an enigma, we have taken steps toward identifying cellular antigens as markers of differentiation. The pressure-resistant SDC in fraction CL that are devoid of the 29.5-kDa protein may be useful for answering questions about the physiological events required for triggering outgrowth and sequential regulation of the Coxiella developmental cycle.

Animals↗

PubMed1996

Immunoelectron microscopy of corticotropes and melanotropes in the pituitary gland of the European ferret, Mustela putorius furo.

The superimposition technique and protein A-gold method of immunoelectron microscopy were employed to study the ultrastructure of corticotropes as well as melanotropes in the ferret pituitary gland. This is the first study ever elucidating the ultrastructural heterogeneity of melanotropes in mammalian pars intermedia on an immunocytochemical basis. Morphological heterogeneity of pars distalis corticotropes has also been elucidated. It is suggested that the different subtypes may represent stages of development (histogenesis), or functional phases of a single cell type. In addition, the occurrence and distribution of both corticotropes and melanotropes in different hypophyseal components have been studied by light-microscopic immunocytochemistry.

Adrenocorticotropic Hormone↗

PubMed1989

Demonstration of intra- and extracellular localization of bullous pemphigoid antigen using cryofixation and freeze substitution for postembedding immunoelectron microscopy.

Using low-temperature postembedding techniques for immunoelectron microscopy, we succeeded in demonstrating the precise localization of bullous pemphigoid antigen (BP-Ag) in normal human skin. Small pieces (less than 1 mm3) of normal adult skin were rapidly frozen in liquid propane at -190 degrees C and subjected to freeze substitution with 100% methanol at -80 degrees C. Specimens were embedded in Lowicryl K11M at -60 degrees C which was polymerized under ultraviolet radiation at -60 degrees C. Ultrathin sections were incubated with BP sera followed by rabbit anti-human IgG and colloidal-gold conjugated anti-rabbit IgG. Epidermal ultrastructure was generally well preserved: the basal cell plasma membrane and intra- and extracellular components of hemidesmosomes could be resolved. Gold particles were mainly distributed on and around the hemidesmosomes in both intra- and extracellular sites, with most of the labelling being inside the basal keratinocytes and within about 300 nm of the basal plasma membrane. No specific labelling was observed beneath melanocytes or when normal human serum was used as a control instead of BP serum. Our observations indicate that BP-Ag is localized in and around hemidesmosomes in normal human skin and that the antigen has both intracellular and extracellular domains with the major component occurring inside the cells.

Autoantigens↗

PubMed1973

Rapid adenovirus typing by immunoelectron microscopy.

A rapid method of typing adenoviruses by immunoelectron microscopy is discribed. This emphasizes the value of an electron microscope in diagnostic virology, especially when a rapid result is required in epidemiology.

Adenoviridae↗

PubMed1980

Immunoelectron microscopy localization of immunoglobulin G in human placenta.

Immunoelectron microscopy of IgG molecules in human mature placenta has shown that IgG bound to microvillar surfaces and the inner wall of endocytotic vesicles of syncytiotrophoblasts. The endocytotic vesicles, containing both bound and unbound IgG molecules, tended to fuse with each other or with other cellular organelles, particularly with lysosomes. The phagolysosomes were more abundant in the basal regions of the cells. Apparently some IgG molecules were not digested by lysosomal enzymes. Vesicles with residual IgG were found to fuse with the basal and basolateral cell membrane and to discharge their contents into the extracellular space by exocytosis. It is suggested that IgG molecules were transported through the trophoblastic basement membrane and the interstitial space by diffusion to the endothelial basement membrane. The IgG molecules then migrated into the fetal vascular lumen via endothelial gaps and interendothelial spaces.

Female↗

PubMed1986

Three types of growth hormone cells of the rat anterior pituitary as revealed by immunoelectron microscopy using a colloidal gold-antibody method.

Immunoelectron microscopy using a colloidal gold-antibody method with anti-rat GH serum demonstrated three morphologically different types of GH cells in the rat anterior pituitary. They were distinguished as Types I, II and III GH cells, containing only large secretory granules about 350 nm in diameter, mixed large and small granules, and only small granules about 150 nm in diameter, respectively. Double gold labeling with large gold particles for GH and small particles for PRL or ACTH indicated that neither GH and PRL nor GH and ACTH were contained in the same cell. In adult male rats, Type I cells (68%) predominated over Type II (22%), while Type III cells were rare (9.7%). On the contrary, in the adult female rats, Type II cells (47%) slightly dominated over Type I (44%) though the rate of Type III cells was the same as in the male. In neonatal infants, the frequency of occurrence of Type III cells was as high as about 20%; sex differences between Types I and II were indistinct. The Type III cells were therefore thought to represent an immature type.

Adrenocorticotropic Hormone↗

PubMed1986

Renal and systemic kappa light chain deposits and their plasma cell origin identified by immunoelectron microscopy.

Kappa light chain determinants were identified by immunoelectron microscopy in nodular glomerulosclerotic lesions and systemic interstitial deposits from a man who died several years after the onset of proteinuric renal failure treated by hemodialysis. He developed adrenal and hepatic failure preterminally but not overt malignant myeloma. Specific labeling was most concentrated over the inner aspect of glomerular basement membrane and the mesangium, which suggested that the protein was nonfiltrable. Tubular basement membrane labeling was densest over the outer aspect, which suggested that the protein perfused from the interstitium rather than from the tubular lumen. We identified the source of the protein as a population of plasma cells present within bone marrow and renal interstitium; these showed specific immunogold labeling for kappa light chain protein over organelles concerned with protein synthesis, secretion, and storage. This appears to be the first identification of light chain determinants in human interstitial para-amyloid deposits with the use of immunogold ultrastructural techniques in tissues prepared for electron microscopy by standard methods and stored as epoxy resin blocks.

Adrenal Glands↗

PubMed1995

Study of neutralizing monoclonal antibodies to bovine herpes virus type-1 (Cooper strain) by immunoperoxidase and immunoelectron microscopy.

Three neutralizing monoclonal antibodies (mAbs) that are specific against bovine herpes virus Type-1 (BHV-1) were studied as to their viral specificity by immunoperoxidase and immunoelectron microscopy. Microscopic examination of GBK BHV-1 infected cells revealed peroxidase activity represented by red-brown granular deposits in the nucleus and cytoplasm. No immunoperoxidase activity was observed in negative controls. For the ultrastructural observations, two approaches were used. Firstly we tested a pre-embedding technique using GBK infected cells, mAbs and gold conjugated-protein A. Gold particles were observed linked to the viral envelopes and to the host cell membrane. Alternatively, a second technique employed BHV-1 purified by potassium tartrate gradients, mAbs and gold conjugated-protein A. After performing the immune reaction, the samples were adsorbed to formvar-coated grids, stained with phosphotungstic acid and observed in a transmission electron microscope. Gold particles were mainly attached to the virion envelope.

Animals↗

PubMedDate not supplied

Localization by immunoelectron microscopy of Schistosoma mansoni antigens in the glomerulus of the hamster (Mesocricetus auratus) kidney.

Adult worm antigen (AWA) and soluble egg antigen (SEA) were localized ultrastructurally by immunoelectron microscopy using two monoclonal antibodies in the glomeruli of hamsters infected with Schistosoma mansoni cercariae or injected with S. mansoni eggs. AWA was detected in all cercaria-infected groups from the 30th day on and was present mainly in cytoplasm of mesangial cells, mesangial matrix, and glomerular basement membrane, either as isolated gold particles or in small electron-dense deposits of probable immune origin. AWA was encountered also on the inner side of the glomerular basal membrane, close to endothelial cells, and in the foot processes of the glomerular epithelial cells. SEA was detected at similar sites, apparently in lesser amounts, in uninfected hamsters inoculated with S. mansoni eggs into the jugular vein. Schistosomal antigens are apparently processed mainly bymesangial cells which are considered to be critical in the pathogenesis of S. mansoni associated glomerulopathy. Mesangioproliferative glomerulonephritis, immunoglobulin (IgG and IgM), and C3 deposits were observed in hamsters in which AWA and SEA were visualized. During early phases of the infection and in hamsters in which granulomatous pneumonitis was induced by S. mansoni eggs, glomeruli were unchanged or showed a slight mesangial proliferation. Our findings suggests that egg antigens also contribute to the pathogenesis of experimental glomerulopathy in the hamster.

Animals↗

PubMed1999

Electron microscopic in situ DNA nick end-labeling in combination with immunoelectron microscopy.

We describe an in situ DNA nick end-labeling method that can be performed at the electron microscopic level and can also be combined with immunoelectron microscopy. As the materials, we used skin tissues from normal skin and from Bowen's disease that had been cryofixed, freeze-substituted, and embedded in Lowicryl K11M resin. Ultrathin sections were cut and incubated with a reaction buffer containing digoxigenin-dUTP and terminal deoxynucleotidyl transferase. Digoxigenin nucleotides were labeled with anti-digoxigenin antibodies conjugated with colloidal gold. Specific signals were detected in the condensed chromatin of differentiated epidermal cells and hair follicles in normal skin and of dyskeratotic cells in Bowen's disease. The labeling density over chromosomal areas of apoptotic cells was significantly higher than that over chromosomal areas of mitotic cells or cytoplasmic areas. Ultrastructure was well preserved and double staining with an anti-keratin antibody was also successfully performed. This simple method has a wide range of applications to identify the nature of apoptotic cells and explore the mechanisms of apoptosis.

Adult↗

PubMedDate not supplied

A new antigen retrieval method using citraconic anhydride for immunoelectron microscopy: localization of surfactant pro-protein C (proSP-C) in the type II alveolar epithelial cells.

In the attempt to develop a pre-embedding immunoelectron microscopic method with improved ultrastructral morphology, we examined an antigen retrieval (AR) method using citraconic anhydride, and compared the effects of glutaraldehyde fixation with routine paraformaldehyde fixation in the pre-embedding immunoelectron microscopic method with reference to the localization of surfactant-associated pro-protein C (proSP-C) in the lung. The glutaraldehyde-fixed tissues were immunostained after AR in 0.05% citraconic anhydride solution, pH 7.4, at 98 degrees C for 60 min. In routine pre-embedding immunoelectron microscopic method using paraformaldehyde fixation, proSP-C positive products were distributed sporadically in the type II alveolar epithelial cells. In glutaraldehyde-fixed tissues without the AR method, proSP-C products were not detected, however after AR in citraconic anhydride proSP-C positive products were distributed specifically, in rough endoplasmic reticulum membranes, Golgi complex membranes, multivesicular bodies and osmiophilic lamellar bodies. The positive proSP-C products also showed lattice-like structures in the alveoli. Thus, the present AR method provides successful pre-embedding immunoelectron microscopy of glutaraldehyde-fixed tissues.

Animals↗

PubMed1991

Immunoelectron microscopy of Fc receptors on the surface of clinical isolates of streptococci.

The distribution of Fc receptors on the surface of clinical isolates of streptococci was examined by immunoelectron microscopy. A unique two-layered ferritin tagging was observed on the surface of isolate MS-4 (group A). The spacing between the layers (10-40 nm) was narrower than that of the layered protein A (30-70 nm) on Staphylococcus aureus. Labeling on the other isolates, IP-28 (group A) and ES-21L (group C), showed similar rough layers, but the labeling were clearly different in thickness and density. The labeling on isolate IP-28 was also similar to that on strain AR1 (group A), reported previously, while the labeling on ES-21L was similar to that on protein G producing strain G148 (group G) in thickness. However, it was obviously thinner than that on IP-28 and AR1. These results strongly suggest that there are at least three distribution patterns of Fc receptors on streptococci, and these are distinct from that of protein A.

Hemagglutination Tests↗

PubMedDate not supplied

A new immunoelectron microscopy approach for the detection of immunoglobulin and complement deposits in epoxy-embedded renal biopsies.

The purpose of this study was to examine the diagnostic value of a new immunoelectron microscopy technique (IEM) for detection of immunoglobulin and complement deposits in epoxy-embedded renal biopsies. Twenty-four renal biopsies were embedded in epoxy resin following a tissue processing involving moderately increased amount of accelerator, DMP-30 (Tri(Dimethyl Amino Methyl) Phenol), in the infiltration steps. Following antigen retrieval by heating in citrate buffer, immunogold labeling was performed on ultrathin sections from these epoxy blocks with antibodies against immunoglobulins and complement. The sections were counterstained with urnayl acetate and lead citrate without any enhancing procedures. The preservation of the ultrastructure with this method was similar to that usually seen in epoxy embedded material. The immunogold labeling was intense and distinct. Immunofluorescence (IF) for light microscopy was carried out on frozen sections of parallel tissue samples. The correspondence between IF and IEM were good, but in some cases higher sensitivity for IgA with IEM than IF was observed in the sense that smaller amounts of antigen were detectable with IEM. The combination of moderately increased amount of accelerator and antigen retrieval is superior to previous methods with respect to ease of use, ultrastructural preservation, and intensity of the immunolabeling. Moreover, the renal tissue can be processed in an automatic ultraprocessor together with other specimens which are to be prepared for routine electron microscopy.

Antigen-Antibody Complex↗

PubMed1977

Immunoelectron microscopy of surface immunoglobulins in pseudolymphoma of the lung. Report of a case.

A case of pseudolymphoma of the lung was studied by light, electron, and immunoelectron microscopy. The tumor consisted predominantly of small lymphoid cells with a few germinal centers. Both light and dark cells were found by electron microscopy. While the dark cells contained abundant free ribosomes, lysosome-like electron dense bodies were more prominent in the light cells. The proportion of surface staining positive pseudolymphoma cells for IgC, IgM, and IgA was high (each about 80%) compared with control spleen cell suspensions by immunoelectron microscopy. But the serum levels of IgG, IgM, and IgA were normal. It is discussed that the polyclonal presence of surface immunoglobulins does not necessarily support the benign nature of the disease.

Adult↗

PubMed1997

Applications of dot-blot, ELISA, and immunoelectron microscopy to field detection of bluetongue virus in Culicoides variipennis sonorensis: an ecological perspective.

An avidin-biotin complex (ABC) dot-blot, an antigen capture enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy (IEM) were used to detect bluetongue (BLU) virus and viral antigen in field-collected C. varriipennis sonorensis Wirth & Jones from an enzootic BLU area in northeastern Colorado. This is the 1st attempt to apply these immunodiagnostic methods to an epidemiologically relevant, large-scale ecological system. One of the 1,800 midges (0.0005%) was positive by the dot-blot procedure, 2 (0.0011%) were positive by the ELISA, and BLU virus was identified in 8 midges (0.0044%) by IEM. These data are interpreted in context of the "whole system" of the disease to provide a framework for determining the knowledge gaps in our understanding and directing future studies in these areas. Our basic model of BLU ecology suggests that the infection rates found by the diagnostic methods are within expected ranges, thus strongly supporting the proposed ecological model and the work used to parameterize the model. This integration of immunodiagnostic methods and ecology makes it evident that further investigations of daily mortality during the extrinsic incubation period are vital to a better understanding of BLU virus occurrence in Culicoides vector and vertebrate host populations.

Animals↗

PubMed1992

Detection of leptospiral antigen (L. interrogans serovar copenhageni serogroup Icterohaemorrhagiae) by immunoelectron microscopy in the liver and kidney of experimentally infected guinea-pigs.

Guinea-pigs were experimentally infected with L. interrogans serovar copenhageni serogroup Icterohaemorrhagiae and their liver and kidney were studied by immunoelectron microscopy using the post embedding indirect immunogold labelling technique. Primary antibody was a purified rabbit anti-serum produced against the same leptospiral strain used in the inoculum. Gold-labelled leptospiral antigen (LAg) was found close to cell membranes of hepatocytes, kidney tubular cells and endothelial cells of the interstitial capillaries of the kidney. Afterwards it was internalized by hepatic and tubular cells, and eventually found in lysosomes. Phagolysosomes of Kupffer cells were also found to contain remnants of degraded leptospires and gold-labelled LAg. Gold-labelled intact leptospires were detected at the enlarged intercellular spaces between hepatocytes at the areas of hepatic cell plate disarray, showing the potential for leptospiral migration during the septicaemic phase of the disease potentially contributing to the pathogenesis of the lesions. The affinity of leptospiral antigenic material for cell membranes suggests an initial interaction with cell surface proteins followed by its internalization and cell damage. The nature of antigenic material detected, however, remains undefined; it may be a toxin, an enzyme or any other factor/s involved in leptospiral virulence.

Animals↗