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PubMed2001

Free-floating cryostat sections for immunoelectron microscopy: Bridging the gap from light to electron microscopy.

Frozen skin sections are routinely used for light microscopic immunohistochemical study of the skin basement membrane zone for two reasons: some skin basement membrane zone proteins are labile to routine chemical fixation, and skin is not amenable to vibratome sectioning. However, inherent limitations of conventional frozen sections, including compromised morphology and a requirement for glass slide-mounting, usually limit immunohistochemical study to the light microscopy level. In the present study, we introduce use of unfixed, free-floating cryostat sections for characterization of immunolocalizations of selected skin basement membrane proteins at both the light and electron microscopy level. The new procedure employs free-floating cryostat sections that can be processed as routine tissue specimens and can be subjected to a variety of special staining procedures including immunohistochemistry. Especially useful is the ease of progressive processing of the same tissue specimen from light microscopy to electron microscopy. In this regard, the method renders itself useful when results of immunolabeling experiments need to be elucidated quickly at histological and ultrastructural levels as required for diagnostic and accelerated investigative strategies.

Antigens, CD↗
PubMed1997

Immunoelectron microscopy shows a clustered distribution of NADPH oxidase components in the human neutrophil plasma membrane.

The NADPH oxidase that produces superoxide in professional phagocytic cells is a flavocytochrome b electron transport chain in the membrane, a heterodimer of gp91phox and p22phox, that is activated by a number of cytosolic proteins, including p47phox, p67phox, and the small GTP-binding protein p21rac, which translocate to the membrane and attach to the flavocytochrome on activation. The components of this oxidase were localized on the cytoplasmic surface of the plasma membrane of adherent unroofed neutrophils by immunolabeling. Components of the NADPH oxidase and p21rac were found together in punctate clusters occupying 0.03-0.1 microm2 of the cytoplasmic surface of the plasma membrane where the density of labeling of the cytosolic components was increased after stimulation with phorbol myristate acetate.

Antibodies, Monoclonal↗
PubMed1992

Localization of the steroid 1-dehydrogenase in Rhodococcus erythropolis IMET 7030 by immunoelectron microscopy.

The steroid 1-dehydrogenase of Rhodococcus erythropolis IMET 7030, an active steroid-transforming strain, was localized by immunogold labelling both in cells induced with 17-alpha-methyl-testosterone and in noninduced cells. The labelling intensity was much higher in induced cells than in noninduced cells, indicating increased enzyme production in the case of induction. Using the postembedding procedure, the main portion of the enzyme was found in the peripheral region of the cytoplasm. A considerable amount was bound in clusters to the inner side of the cytoplasmic membrane. The enzyme was also detected in channels connecting the cytoplasm with the cell surface. By means of the preembedding labelling, a few gold clusters could be detected on the cell surface. The significance of this observation was discussed.

Cytoplasm↗
PubMed1993

Further approaches to the quaternary structure of octopus hemocyanin: a model based on immunoelectron microscopy and image processing.

The direction of the polypeptide chains and the location of the functional units in Octopus vulgaris hemocyanin were studied by various methods. Monoclonal antibodies specific for the Ovc (clone Ov409) and Ovg (clone Ov315) functional units produced immunocomplex strings which were examined in the electron microscope. In both cases the immunocomplexes contained more than two hemocyanin molecules in their side view, demonstrating that in the whole hemocyanin neighboring polypeptide chains run in antiparallel directions. The interhemocyanin distances in the immunocomplexes also indicated that Ovg is located inside the cylinder, while Ovc is located in the external layers of functional units. In addition, the fact that the binding point of the Fab arm to the hemocyanin molecule was occasionally visible confirmed the external location of functional unit Ovc. Image processing of the whole hemocyanin cross-linked with dimethyl suberimidate showed that the end-on view is not a perfect cylinder but a regular pentahedron and that the five-arch collar is probably composed of five pairs of functional unit Ovg located inside the cylinder. The accessibility of cross-linked hemocyanin to functional unit-specific polyclonal antibodies, studied in immunoelectrophoresis, showed that Ovb and Ove are highly accessible, while Ovd, Ovf, and Ovg are not. The low accessibility of Ovd may be at least partially explained by its high sugar content which could hamper the accessibility of the antibody to the antigen.

Animals↗
PubMed1995

Intranuclear distribution of galectin-3 in mouse 3T3 fibroblasts: comparative analyses by immunofluorescence and immunoelectron microscopy.

The intracellular distribution of carbohydrate binding protein 35 (CBP35), recently named galectin-3, was studied in mouse 3T3 fibroblasts, using immunofluorescence at the light microscope level and immunogold labeling at the ultrastructural level. In general, serum-stimulated, proliferating cells showed higher levels of labeling than quiescent cultures of the same cells. In the proliferating cells, the labeling intensity was higher in the nucleus than in the cytoplasm. Treatment of permeabilized cells or thin sections with ribonuclease A decreased the immunolabeling intensity, whereas parallel control treatments with deoxyribonuclease I failed to yield the same effect. While there appears to be general agreement between the immunofluorescence and the ultrastructural results regarding the level of CBP35 and its association with nuclear ribonucleoprotein complexes, there was one striking difference in terms of labeling of specific subnuclear structures. Immunofluorescence results indicate diffuse distribution of CBP35 within the nucleus, but the label appears to be excluded from certain "black holes," which most probably correspond to nucleoli. On the other hand, immunogold particles were observed in electron microscopy, mainly in interchromatin spaces, except for interchromatin granule clusters, at the border of condensed chromatin, on the dense fibrillar component, and at the periphery of the fibrillar centers of nucleoli.

3T3 Cells↗
PubMed2000

Immunoelectron microscopy provides evidence that tumor necrosis factor receptor-associated protein 1 (TRAP-1) is a mitochondrial protein which also localizes at specific extramitochondrial sites.

The tumor necrosis factor receptor-associated protein 1 (TRAP-1) interacts with a variety of proteins involved in diverse functions. We have used quantitative immunogold electron microscopy and biochemical analysis to evaluate the subcellular distribution of TRAP-1 in rat tissues. Immunofluorescence employing a polyclonal antibody raised to human recombinant TRAP-1 reveals specific staining of mitochondria and nuclear region in mammalian cells. Western blot analysis of purified rat liver mitochondrial subfractions with the TRAP-1 antibody reveals that the cross-reactive protein (M(r) approximately 80 kDa) is mainly present in the matrix compartment. Immunogold labeling of rat tissue sections embedded in LR Gold resin shows strong labeling of mitochondria in all the tissues examined (viz., liver, heart, pancreas, kidney, spleen, anterior pituitary gland). Additionally, specific and significant labeling with TRAP-1 antibody was also observed in certain tissues in a number of nonmitochondrial locations, including pancreatic zymogen granules, insulin secretory granules, cardiac sarcomeres, and nuclei of pancreatic and heart cells, and on the cell surface of blood vessel endothelial cells. Western blot analysis showed that a cross-reactive protein of similar molecular mass as TRAP-1 is present in purified pancreatic zymogen granules. Immunogold labeling was prevented in all tissues by preadsorption of the TRAP-1 antibody with the purified recombinant TRAP-1 protein. These observations and the fact that TRAP-1 is synthesized with a typical mitochondrial targeting presequence strongly indicate that TRAP-1 is primarily a mitochondrial matrix protein. The localization of this protein at specific extramitochondrial sites raises interesting and fundamental questions regarding the possible mechanisms by which these proteins are translocated to such sites.

Animals↗
PubMedDate not supplied

Three-dimensional immunoelectron microscopy of scorpion hemocyanin labeled with a monoclonal Fab fragment.

An immunocomplex of the 4 x 6-meric hemocyanin of the scorpion Androctonus australis with the monoclonal Fab fragment L104 was reconstructed from electron micrographs of a negatively stained specimen, using the double-carbon-layer technique. The resulting structure enables a clear visualization of the Fab fragments bound to the four copies of the Aa6 subunit and directly confirms a previous localization of the L104 epitope deduced from two-dimensional image processing. Despite a strong flattening effect produced by the negative-staining technique the orientations of the Fab fragments are well characterized. Moreover, the observation of a central hole within the elbow bends of the Fab fragments provides information about the disposition of the Fabs around their main axis.

Animals↗
PubMed2001

Immunoelectron microscopy in embryos.

Immunogold labeling of proteins in sections of embryos embedded in acrylate media provides an important analytical tool when the resolving power of the electron microscope is required to define sites of protein function. The protocol presented here was established to analyze the role and dynamics of the activated protein kinase C/Rack1 regulatory system in the patterning and outgrowth of limb bud mesenchyme. With minor changes, especially in the composition of the fixative solution, the protocol should be easily adaptable for the postembedding immunogold labeling of any other antigen in tissues of embryos of diverse species. Quantification of the labeling can be achieved by using electron microscope systems capable of supporting digital image analysis.

Animals↗
PubMed1990

Associations between beta-tubulin and mitochondria in adult isolated heart myocytes as shown by immunofluorescence and immunoelectron microscopy.

We have investigated the associations between beta-tubulin and mitochondria in freshly isolated cardiac myocytes from the rat. Beta-tubulin was identified by using monoclonal antibodies for immunofluorescence and high resolution immunogold electron microscopy. In addition, conventional transmission and scanning electron microscopic studies were performed. After chemical stabilization in a formaldehyde solution, the myocytes were shock-frozen at -150 degrees C, cryosectioned at -70 degrees C and subsequently processed for immunohistochemical and immunocytochemical microscopy. A characteristic of the rod shaped myocytes is the presence of a dense network of microtubules in the cytoplasm displaying a pattern of strong anti-beta-tubulin reaction. The complexity of this network however varies considerably among the myocytes reflecting microtubule dynamic instability. Further, our findings demonstrate that the beta-tubulin label in rod cells is confined to the perinuclear and interfibrillar spaces and, therefore, is largely colocalized with the cytoplasmic organelles. In myocytes undergoing severe contracture the distribution of beta-tubulin is entirely restricted to the outer mitochondrial-containing domain. This implies that, in a cell model with marked segregation of the contractile filaments and organelles, mitochondria are codistributed with microtubules in the total absence of desmin intermediate filaments. Moreover, our immunogold preparations demonstrate anti-beta-tubulin labelling in the outer mitochondrial membrane as well as of fibres in close apposition to this membrane. These results indicate the presence of a specific beta-tubulin binding to the outer mitochondrial membrane that probably also involves microtubule based translocators and/or MAPs.

Animals↗
PubMed1995

DNA localization in nuclear fragments of apoptotic ameloblasts using anti-DNA immunoelectron microscopy: programmed cell death of ameloblasts.

Ameloblasts responsible for tooth enamel formation are classified into two different phases: secretion and maturation. At the transition between these secretion and maturation stages, a considerable number of cells die. In this study, we examined the morphology of degenerating ameloblasts by conventional electron microscopy, and DNA cleavage in degenerating ameloblast nuclei by the in situ terminal transferase assay. The results suggest that apoptosis (programmed cell death) in ameloblasts, including DNA ligation is induced at the transitional stage. The nuclear fragments, chromatin condensation and DNA relocation in apoptotic nuclei were examined quantitatively by post-embedding anti-DNA immunogold electron microscopy and the in situ terminal transferase assay combined with electron microscopy. Numerical analysis revealed that immunogold labeling density in the condensed chromatin of apoptotic nuclei was comparable on the average to that in the perinuclear heterochromatin of normal nuclei, and that individual apoptotic nuclear fragments exhibited highly variable to that of normal heterochromatin, to fragments with densities twice as high as that of normal heterochromatin. The in situ terminal transferase assay combined with electron microscopy detected DNA ends exposed by ultrathin sectioning as well as DNA cleavage by a putative endonuclease. In conclusion, the state of the DNA, including its ligation and degeneration, changes gradually during chromatin condensation and nuclear fragmentation of apoptosis.

Ameloblasts↗
PubMed1996

Pre- and post-embedding immunoelectron microscopy of Ki-M1P immunoreactive germinal center macrophages using ultra-small gold probes with silver enhancement.

The Ki-M1P protein is primarily detected in cells deriving from the monocyte/macrophage cell lineage. The aim of this study was to investigate the occurrence of Ki-M1P immunoreactivity in germinal center macrophages by immunohistochemical and immunocytochemical staining techniques. Ultra-small (0.8 nm) gold probes combined with silver enhancement were used as a detection system for pre- and post-embedding immunostaining both at the light and electron microscopic level. Ki-M1P-positive macrophages were observed at a constant frequency in the germinal centers of the follicles throughout the tonsillar lymphatic tissue. The specific immunostaining was localized in the cytoplasm of these cells. Electron microscopic examination demonstrated the presence of abundant lysosomes in the cytoplasm, and some of the germinal center macrophages contained phagocytosed cells (tingible bodies) showing signs of various degrees of digestion. Ki-M1P immunoreactivity, as revealed by depositions of silver-enhanced ultra-small gold probes, was confined to the periphery of the lysosomes and tingible bodies. The results obtained demonstrate that the use of silver-enhanced ultra-small gold probes is a highly sensitive and specific detection system for pre- and post-embedding immunostaining of the Ki-M1P protein, and provides, in general, a flexible system for combined light and electron microscopic examination of tissue antigens. Furthermore, in the cytoplasm of the germinal center macrophages a spatial association between the Ki-M1P protein and lysosomes and tingible bodies was observed. These findings may indicate that the Ki-M1P protein is connected with phagocytosis and/or processes related to intracellular digestion in these cells.

Antibodies, Monoclonal↗
PubMed1993

The impact of aging on enzyme proteins of rat liver peroxisomes: quantitative analysis by immunoblotting and immunoelectron microscopy.

The alterations of hepatic peroxisomes and their enzymes during aging were investigated in male rats. Peroxisomes in the livers of young (2 months) and old (39 months) male Wistar rats were analyzed by morphometry and quantitative immunocytochemistry, as well as by immunoblotting of highly purified peroxisomal fractions. Immunoblots showed that catalase and acyl-CoA oxidase were decreased in peroxisomes of old animals but the trifunctional enzyme, thiolase, and urate oxidase were increased. The morphometrical analysis revealed a heterogeneous distribution of peroxisomes in the liver lobule of the old animals, with a significant elevation of peroxisomal volume density in pericentral over periportal hepatocytes, in contrast to the uniform pattern in the young rats. Furthermore, age-related lobular gradients were also observed by quantitative immunocytochemistry in the peroxisomal concentrations of trifunctional enzyme (central > portal) and, inversely, for catalase (portal > central). Whereas acyl-CoA oxidase was diminished across the liver lobule, the enzyme 3-ketoacyl-CoA thiolase was elevated. These observations show that peroxisomes are significantly altered in aged animals and suggest that these alterations may contribute to the disturbance of lipid metabolism in aged animals. Moreover, the diminution in catalase and the elevation of urate oxidase could contribute to the oxidative stress which is considered to be of fundamental importance in the aging process.

Acetyl-CoA C-Acetyltransferase↗
PubMed1998

Characterization of Aspergillus nidulans peroxisomes by immunoelectron microscopy.

In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies (peroxisomes) in Aspergillus nidulans. Although at a lower level, proliferation of peroxisomes also occurs in cells growing under conditions that induce penicillin biosynthesis. Here, microbodies in oleate-grown A. nidulans cells were characterized by using several antibodies that recognize peroxisomal enzymes and peroxins in a broad spectrum of eukaryotic organisms such as yeast, and plant, and mammalian cells. Peroxisomes were immunolabeled by anti-SKL and anti-thiolase antibodies, which suggests that A. nidulans conserves both PTS1 and PTS2 import mechanisms. Isocitrate lyase and malate synthase, the two key enzymes of the glyoxylate cycle, were also localized in these organelles. In contrast to reports of Neurospora crassa, our results demonstrate that A. nidulans contains only one type of microbody (peroxisomes) that carry out the glyoxylate cycle and contain 3-ketoacyl-CoA thiolase and proteins with the C-terminal SKL tripeptide.

Animals↗
PubMed2001

Immunoelectron microscopy provides evidence for the presence of mitochondrial heat shock 10-kDa protein (chaperonin 10) in red blood cells and a variety of secretory granules.

Hsp10 (10-kDa heat shock protein, also known as chaperonin 10 or Cpn10) is a co-chaperone for Hsp60 in the protein folding process. This protein has also been shown to be identical to the early pregnancy factor, which is an immunosuppressive growth factor found in maternal serum. In this study we have used immunogold electron microscopy to study the subcellular localization of Hsp10 in rat tissues sections embedded in LR Gold resin employing polyclonal antibodies raised against different regions of human Hsp10. In all rat tissues examined including liver, heart, pancreas, kidney, anterior pituitary, salivary gland, thyroid, and adrenal gland, antibodies to Hsp10 showed strong labeling of mitochondria. However, in a number of tissues, in addition to the mitochondrial labeling, strong and highly specific labeling with the Hsp10 antibodies was also observed in several extramitochondrial compartments. These sites included zymogen granules in pancreatic acinar cells, growth hormone granules in anterior pituitary, and secretory granules in PP pancreatic islet cells. Additionally, the mature red blood cells which lack mitochondria, also showed strong reactivity with the Hsp10 antibodies. The observed labeling with the Hsp10 antibodies, both within mitochondria as well as in other compartments/cells, was abolished upon omission of the primary antibodies or upon preadsorption of the primary antibodies with the purified recombinant human Hsp10. These results provide evidence that similar to a number of other recently described mitochondrial proteins (viz., Hsp60, tumor necrosis factor receptor-associated protein-1, P32 (gC1q-R) protein, and cytochrome c), Hsp10 is also found at a variety of specific extramitochondrial sites in normal rat tissue. These results raise important questions as to how these mitochondrial proteins are translocated to other compartments and their possible function(s) at these sites. The presence of these proteins at extramitochondrial sites in normal tissues has important implications concerning the role of mitochondria in apoptosis and genetic diseases.

Animals↗
PubMedDate not supplied

Tubules of the trans Golgi apparatus visualized by immunoelectron microscopy.

Tubules constitute an integral part of the Golgi apparatus and have been shown to form a complex and dynamic network at its trans side. We have studied in detail structural features of the trans Golgi network and its relationship with the cisternal stack in thin sections of Lowicryl K4M embedded human absorptive enterocytes by immunolectron microscopy. Immunoreactive sites for alpha1,3 N-acetylgalactosaminyltransferase and blood group A substance were detectable throughout the cisternal stack and the entire trans Golgi network. Furthermore, the entire trans Golgi network was reactive for CMPase activity. Evidence for two kinds of tubules at the trans side of the Golgi apparatus was found: tubules that laterally connect adjacent and distant cisternal stacks, and others extending from central and lateral portions of trans cisternae to form the complex and extensive trans Golgi network. Trans cisternae showed often the peeling-off phenomenon and were continuous with the trans Golgi network. Both, trans cisternae and tubules of the trans Golgi network exhibited regionally buds and vesicles with a lace-like, non clathrin coat, previously reported by others in NRK cells, which contained glycoproteins with terminal N-acetylgalactosamine residues. These buds and vesicle are therefore involved in constitutive exocytosis.

Acid Phosphatase↗
PubMed1994

Immunoelectron microscopy of enkephalinergic innervation of GABAergic neurons in the periaqueductal gray.

The pre-embedding double immunoreaction method was used to study synaptic relations of enkephalinergic and GABAergic neuronal elements in the ventrolateral part of the periaqueductal gray of the Wistar albino rat. The enkephalin-like neuronal elements were immunoreacted by the silver-gold intensified peroxidase-antiperoxidase method and the GABA-like immunoreactive neurons were immunoreacted by the unintensified peroxidase-antiperoxidase method. GABA-like immunoreactive neuronal somata were post-synaptic to both the enkephalin-like immunoreactive and the non-immunoreactive axon terminals. Enkephalin-like immunoreactive axon terminals were found to make synapses with GABA-like immunoreactive and non-immunoreactive dendrites. The synapses between the two kinds of chemically characterized neurons appeared to be both asymmetrical and symmetrical. Possible functional activity related to pain modulation, and synaptic relations between the enkephalinergic and GABAergic neurons in the periaqueductal gray and the dorsal raphe nucleus, are discussed.

Animals↗
PubMed1995

Immunoelectron microscopy of beta-endorphinergic synaptic innervation of nitric oxide synthase immunoreactive neurons in the dorsal raphe nucleus.

On the basis of the comparing of the distribution of beta-endorphin-like immunoreactive neuronal fibres and nitric oxide synthase-like immunoreactive neurons in the dorsal raphe nucleus, the synapses between the two immunocytochemically identified neurons were studied with a modified DAB-silver-gold intensification double immunostaining technique at the electron microscopic level. Although both of them can be found in the mediodorsal and medioventral parts of the dorsal raphe nucleus, the synapses between them could only be found in the mediodorsal part. The majority of the beta-endorphin-like immunoreactive neuronal fibers contained many dense-cored vesicles. The synapses made by beta-endorphin-like immunoreactive neuronal axon terminals on nitric oxide synthase-like immunoreactive neurons were both symmetrical and asymmetrical with the former predominant, especially in the axo-dendritic ones. beta-Endorphin-like immunoreactive perikarya could only be found in the ventrobasal hypothalamus. These findings suggest the possibility that the beta-endorphin- producing neurons in the ventrobasal hypothalamus could influence nitric oxide synthase-containing neurons in the dorsal raphe nucleus by synaptic relations.

Animals↗