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PubMedDate not supplied

Immunoelectron microscopy on material retrieved from paraffin: accurate sampling on the basis of stained paraffin sections.

As a rule, immunoelectron microscopy (immuno-EM) is performed on fresh material processed according to specialized methods (e.g., freezing or embedding in hydrophilic resins). Paraffin-embedded tissue has only occasionally been used as a source of material for immuno-EM; this was usually as a last resort, when no fresh material was available. The authors used archival formalin-fixed, paraffin-embedded basal cell carcinomas for studying the fine-structural distribution of the cell surface molecule CD44 and its variants, as well as some other antigens. The results demonstrate, firstly, that paraffin-embedded material is far more suitable for immuno-EM than frequently assumed and, secondly, that the use of paraffin-embedded material enables highly accurate sampling on the basis of immunohistochemically or conventionally stained light microscopic sections.

Basal Cell Carcinoma↗

PubMed1985

An enteric coronavirus of the rabbit: detection by immunoelectron microscopy and identification of structural polypeptides.

The immunoelectron microscopy (IEM) technique has been used for the detection of a rabbit enteric coronavirus (RECV). Immune serum was prepared in guinea pigs; the viral antigen used for the immunization procedure was obtained from the caecum of a sick rabbit, concentrated by centrifugation and purified on Percoll gradient. In order to identify the viral particles used in the immunization procedure, the protein pattern of the particles was determined by electrophoresis and compared with the pattern of other known coronaviruses. Analysis of structural polypeptides of the purified viral particles revealed a pattern similar to that reported for other coronaviruses. These polypeptides cross reacted with two other coronavirus specific immune sera (IBV and TGE). IEM assay of fecal samples collected from healthy and sick rabbits showed the presence of immune aggregates in specimens from both sick and healthy rabbits. Those aggregates contained viral particles sharing morphological characteristics with other coronaviruses. Furthermore, IEM assay was shown to be more sensitive than a direct EM procedure to detect coronavirus particles in rabbit feces. This assay also allowed the detection of a larger number of chronic carriers.

Animals↗

PubMed1985

Projection of olfactory bulb efferents to layer I GABAergic neurons in the entorhinal area. Combination of anterograde degeneration and immunoelectron microscopy in rat.

Glutamic acid decarboxylase (GAD) immunoelectron microscopy in combination with anterograde degeneration was applied in rats to study the synaptic targets of olfactory bulb afferents to the lateral subdivision (LEA) of the entorhinal area (EA). Immunoreactive neurons and terminals are scattered throughout all layers of LEA. After olfactory bulb resection, terminal degeneration occurs in layer Ia of EA. Using the electron microscope we examined serial thin sections of 12 and 14 immunoreactive neurons sampled from layer Ia of the dorsal (DLEA) and ventral (VLEA) subdivisions of LEA, respectively. The morphology of all these neurons is similar: they are small (short axis 5-9 micron, long axis 7-12 micron) and possess eccentrically located, indented nuclei provided with filamentous nuclear rodlets. The immunoreactive neurons have thin, smooth dendrites which usually emerge abruptly from the somata. We observed a single cilium on 5 of the immunoreactive neurons. In layer Ia of both DLEA and VLEA, the somata of the immunoreactive neurons are contacted by degenerating, non-immunoreactive boutons showing asymmetric synaptic junctions. In addition to these boutons, 4 other categories of axo-somatic terminals can be distinguished: normal, non-immunoreactive boutons forming asymmetric synapses and containing spherical synaptic vesicles; normal, non-immunoreactive boutons with symmetric synapses and pleomorphic synaptic vesicles; normal, non-immunoreactive boutons with asymmetric synapses, containing dense-cored vesicles in addition to spherical synaptic vesicles; and normal, immunoreactive boutons with symmetric synapses and pleomorphic synaptic vesicles. It is suggested that the GAD-immunoreactive neurons which receive olfactory bulb input correspond to local circuit neurons with intralaminar axons which innervate each other as well as the distal segments of the apical dendrites of projection neurons with cell bodies in layers II and III. Thus, the olfactory input in EA seems to be wired not only for excitation of layers II and III pyramidal neurons but also for feed-forward inhibition using GABAergic intermediary neurons, strategically located in the area of termination of olfactory bulb fibers.

Animals↗

PubMed1988

Calcitonin and chromogranin A localization in medullary carcinoma of the thyroid by immunoelectron microscopy.

We used a post-embedding immunoelectron microscopy method, using protein A-gold, to detect calcitonin and chromogranin A immunoreactivity in three cases of human medullary thyroid carcinoma. Because the epoxy-embedded tissue had been fixed (glutaraldehyde or formaldehyde) and osmicated before embedment, the proteins were identified in optimally preserved tissue. Uranyl and lead staining was used after immunolabeling, so that the tissue was also optimally contrasted. The morphological advantage provided by osmication was tested by labeling rat thyroid gland C-cells for calcitonin. The protein A-gold technique allowed localization of both antigens to the contents of membrane-bound secretory granules in the tumor cells. In one case, labeling density for each antigen was measured over several intercellular compartments and the interstitium. Calcitonin, but not chromogranin A, reactivity was also identified in intracellular amyloid fibrils in two cases, showing that the constant region of calcitonin is preserved in amyloid deposits related to the tumor cells.

Calcitonin↗

PubMed2001

Quantitative immunoelectron microscopy of type VI collagen in glomeruli in type I diabetic patients.

BACKGROUND: The nature of the extracellular matrix (ECM) accumulating in the glomerular basement membrane (GBM) and mesangium (Mes) in diabetes is unknown. Type IV collagen (Col IV) as estimated by quantitative immunoelectron microscopy was reduced in type I diabetic patients (D) with rapid ("fast-track") compared with slow ("slow-track") development of diabetic nephropathy (DN) lesions and controls (C). Col VI is another ECM component suggested to account for Mes matrix (MM) expansion in DN. METHODS: Col VI ECM density was evaluated in eight "slow-track" {Mes fractional volume [Vv(Mes/glom)] <0.32, duration> 20 years} and seven "fast-track" patients [Vv(Mes/glom)> 0.37, duration <20 years diabetes] and in eight C. Quantitative immunoelectron microscopy was performed using polyclonal antibodies to Col VI. Gold particle density (PDG) in MM and the inner layer (IL) of the GBM was measured using stereologic methods. RESULTS: GBM IL PDG was decreased in both fast-track (1.7 +/- 1.6/microm2, mean +/- SD, P < 0.002) and slow-track (3.9 +/- 2.4, P < 0.02) D versus C (10.8 +/- 7.9). GBM IL PDG was also lower in the fast-track versus slow-track D (P < 0.04). Mes matrix PDG/microm2 was decreased in fast-track D (3.2 +/- 3.6) versus C (14.1 +/- 14.6, P < 0.02); a similar trend was seen in slow-track D (5.7 +/- 5.6, P < 0.1). There was no significant difference in MM PDG between the slow-track and fast-track D. CONCLUSION: Col VI density in MM and GBM is decreased in diabetic patients with slowly and rapidly developing renal lesions. This leaves the nature of ECM accumulation in DN unexplained. At least in part, glomerular ECM compositional change is related to diabetes per se and may be independent of the severity of lesions.

Adult↗

PubMed1992

Restriction of cicatricial pemphigoid antigens to the lamina densa: confirmation by indirect immunoelectron microscopy.

Circulating anti-basement membrane zone (BMZ) antibodies in a patient with cicatricial pemphigoid (CP) were examined using an indirect immunofluorescence test, indirect immunoperoxidase electron microscopy, and Western blot analysis. An indirect immunofluorescence test on salt-split skin revealed that the anti-BMZ antibodies reacted solely to the dermal side at the separating epidermal-dermal interface, and indirect immunoelectron microscopy on intact skin indicated localization of the corresponding antigens (CP antigens) over the lamina densa and within the lower half of the lamina lucida; there were no CP antigens beneath a melanocyte. Indirect immunoelectron microscopy on salt-split skin demonstrated that the CP antigens were partly dissociated from, but restricted to, the lamina densa. Western blot analysis showed no differences in molecular weight between the CP antigens and bullous pemphigoid (BP) antigens. CP antigens, as detected by this patient's serum, appear to be constituted of molecules quite similar to BP antigens, but with different epitopes. CP antigens may be shed from basal cells and locate in the area of anchoring filaments, where they play a role in connecting basal cells to the underlying lamina densa.

Autoantibodies↗

PubMed1995

Immunoelectron microscopy and epitope mapping with monoclonal antibodies suggest the existence of an additional N-terminal transmembrane helix in the cytochrome b subunit of bacterial ubiquinol:cytochrome-c oxidoreductases.

The topology of the ubiquinol:cytochrome-c oxidoreductase (cytochrome bc1 complex) from Paracoccus denitrificans was investigated by immunoelectron microscopy with sequence-specific murine monoclonal antibodies. Epitope mapping with synthetic peptides and enzymic proteolytic cleavage of the cytochrome bc1 complex were employed to localize precisely the respective antibody epitopes on the subunits of this membrane protein complex. Localization of defined epitopes on the cytochrome bc1 complex by immunoelectron microscopy clearly demonstrates that the N-terminus of the cytochrome b subunit is exposed to the periplasmic space. This finding is in agreement with a nine-transmembrane-helices topology model (I-IX) as predicted before for cytochrome b. However, due to other published evidence we favour the existence of an additional transmembrane helix (helix 0) complementing a more recently published eight-helices model (A-C,cd, D-H), at least for prokaryotes.

Amino Acid Sequence↗

PubMed1983

A human monoclonal antibody reacting with Merkel cells: immunofluorescence, immunoperoxidase, and immunoelectron microscopy.

A human monoclonal, mu, kappa, cold agglutinin antibody of the rare specificity Pr h (serum and proper eluates) was used in immunofluorescence and immunoperoxidase techniques and in immunoelectron microscopy on rabbit lip specimens. Pr h antibody strongly reacted with scattered cells in epidermis, which were demonstrated to be Merkel cells by electron microscopy; no nerve fibers were stained. In immunoelectron microscopy (IEM), a strong reaction was seen within the cytoplasm and around the granules. This is the first IEM staining of Merkel cells (MC) so far reported; it demonstrates the expression of a carbohydrate differentiation antigen in MC. The availability of a potent monoclonal antibody reacting with MC but not with neighboring epidermal cells in rabbit lip offers a new tool for the study of several aspects of MC biology, including antigenic properties and kinetics.

Animals↗

PubMed2001

Expression of cathepsin K mRNA and protein in odontoclasts after experimental tooth movement in the mouse maxilla by in situ hybridization and immunoelectron microscopy.

This study demonstrated the simultaneous expression of cathepsin K (CK) mRNA by in situ hybridization and CK protein by immunoelectron microscopy in odontoclasts in mouse maxillae after experimental tooth movement. On the pressure side (the area under pressure during tooth movement), CK mRNA was detected in odontoclasts in resorption lacunae in the tooth root, in osteoclasts in bone resorption lacuane, and in fibroblasts in the periodontal ligament. Using electron microscopy, CK protein was detected at the apex of odontoclasts, intracellularly in vesicles and granules, and extracellularly in irregularly shaped vacuoles (extracellular spaces), on the plasma membrane of the ruffled border, and on and between typical striated type I collagen fibrils in the lacunae. These vesicles and granules appeared to fuse with irregular vacuoles containing CK-positive fragmented fibril-like structures close to the ruffled border. In the basolateral portion of odontoclasts, small amounts of CK-positive rough endoplasmic reticulum (ER) were found. CK-positive intracellular vacuoles (not extracellular spaces) also appeared to fuse with the vesicles and granules. However, these fused organelles rarely contained fragmented fibril-like structures. They are probably endolysosomes. The distribution of CK in odontoclasts was similar to that previously seen in osteoclasts. Furthermore, CK-positive fibril-like structures were found in the vacuoles of fibroblasts. These results indicated that during tooth movement CK is synthesized in odontoclasts on the pressure side and secreted into the tooth resorption lacunae. Therefore, CK may take part in the degradation of the dentin matrix (type I collagen fibrils and non-collagenous protein) of the tooth root, and in the subsequent intracellular degradation of endocytosed fragmented fibril-like structures in endolysosomes.

Animals↗

PubMedDate not supplied

Localization of a sequence motif complementary to the nuclear localization signal in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy.

A sequence motif complementary to the nuclear localization signal (NLS) has been localized in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy using sequence-specific antibodies. The antibodies were generated in two different ways: by immunization with a carrier-coupled peptide and by isolation of the sequence-specific antibody from an immune serum against native proteasomes using a peptide-affinity column. The sequence specificity of the isolated antibody was confirmed by a PEPSCAN-ELISA performed on overlapping nonapeptides deduced from the sequence of the alpha-subunit of the Thermoplasma proteasome. Compared to the antibody induced by the carrier-coupled peptide this antibody fraction showed a much higher affinity for native proteasomes. The attachment site of the Fab portion of the antibody to the proteasome was mapped by electron microscopy in conjunction with image processing. The antibody was found to bind to the periphery of the two outer "disks" of the proteasome complex formed by the alpha-subunits.

Amino Acid Sequence↗

PubMed1997

Bluetongue virus in laboratory-reared Culicoides variipennis sonorensis: applications of dot-blot, ELISA, and immunoelectron microscopy.

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 or viral antigen or both in adult Culicoides variipennis sonorensis Wirth & Jones. The dot-blot and ELISA procedures detected viral antigen in 10-22% (depending on serotype) of the biting midges infected with BLU-2, BLU-10, BLU-13, and BLU-17 and approximately 68% of the midges infected with BLU-11. IEM analyses revealed BLU virus in salivary glands, fat body, and thoracic muscle tissue from infected insects. There appeared to be selective growth of the virus in salivary gland tissue.

Animals↗

PubMed1991

Technique for transmission and immunoelectron microscopy of the human endolymphatic sac and vestibular epithelia.

A surgical technique is presented to obtain the entire vestibular aqueduct, containing the endolymphatic duct and sac as well as the vestibular epithelia, from the maculae and cristae during labyrinthectomy. The inner ear tissue was fixed in 3% glutaraldehyde, decalcified in 0.1 mol/L Na-EDTA, and routinely processed for transmission electron microscopy, including post-fixation with osmium tetroxide. Postembedding protein A-colloidal gold immunoelectron microscopy was performed after exposure of cellular antigenic sites by sodium metaperiodate. To validate this technique, thin sections from the crista ampullaris and the endolymphatic sac were incubated with antibodies raised against S-100, a protein present in specific types of neural tissue. Specific immunoreactivity was observed in the crista ampullaris, but not in the endolymphatic sac. The surgical biopsy technique described provides a means for the neuro-otologist to collect inner ear tissue from surgical patients that can be used for pathological studies using immunohistochemistry and electron microscopy.

Biopsy↗

PubMed1990

Effect of resin use in the post-embedding procedure on immunoelectron microscopy of membranous antigens, with special reference to sensitivity.

To investigate quantitatively the effect of resins on the sensitivity of immunoelectron microscopy of membranous antigen, ultra-thin sections of bovine epithelial tissue embedded in five different kinds of resins [JB-4 (JB4), LR Gold (LRG), Lowicryl K4M (K4M), Quetol 812 (Q812), and Spurr's (Spurr) resin] were labeled specifically with anti-desmosomal glycoprotein I(DGI) antibody followed by protein A-gold (PAG) conjugates. When we compared the labeling intensity expressed as the number of PAG particles per 500-nm length of the desmosomal region along the membrane, three hydrophilic resins (JB4, LRG, and K4M) showed much greater levels of labeling intensity than did epoxy resins (Q812 and Spurr), which had a negative value. The three hydrophilic resins showed only minor differences in their levels of labeling intensity. The intensity obtained with JB4, which was the highest of the three, was further increased by pretreatment of the ultra-thin sections with methyl methacrylate monomer (MM) for 5 min. On the basis of these results, wide applicability of this new technique for membranous antigens, which have been difficult to detect positively by any previously employed techniques, is suggested.

Animals↗

PubMed1996

A comparison of methods for heat-mediated antigen retrieval for immunoelectron microscopy: demonstration of cytokeratin No. 18 in normal and neoplastic hepatocytes.

Postembedding antigen retrieval is a well established technique for immunoelectron microscopy; however, many antigens cannot be detected without additional unmasking procedures. This study was undertaken to determine whether microwave oven heating, autoclaving, and pressurized boiling, which are well recognized methods of antigen retrieval for light microscopy, and simple boiling can also be used in electron microscopy. We investigated neoplastic and normal hepatocytes using a commercially available mouse monoclonal antibody against cytokeratin NO. 18 (CK 18). The tissue was fixed in paraformaldehyde/glutaraldehyde and embedded in Lowicryl K4M at -40 C. Ultrathin sections in various buffers were exposed to heat using one of four methods or to pronase at 37 C before incubation with the primary antibody. The secondary antibody was gold-labeled goat anti-mouse antibody. Sections that were not heat-treated remained unlabeled, but heat-treated sections showed immunoreactivity located mainly at the cytoplasmic periphery. Some of the gold particles lay in direct or loose association with intermediate filaments, some were seen in the area of desmosomes, and some did not appear related to any structures. No difference in immunostaining was found among the four methods of heat treatment. The citrate buffer, pH 6.0, and 10 mM EDTA, pH 8.0, generated the best labeling results.

Antigens↗

PubMedDate not supplied

Ultrastructural study of a pituitary adenoma (prolactinoma) within the clivus bone using immunoelectron microscopy.

In a case of a pituitary adenoma in the clivus bone in a 71-year-old man, ultrastructural investigation using conventional aldehyde-fixed, epoxy-embedded tissue revealed the tumor to be composed of cells with euchromatic nuclei, dense nucleoli, abundant rough endoplasmic reticulum, spherical secretory granules, and granule extrusion at the lateral cell surface, all of which suggest a prolactin-producing adenoma. Using a protein A-gold immunolabeling technique on snap-frozen tissue subsequently fixed in a mild fixative and embedded in a hydrophilic resin, the presence of prolactin immunoreactivity within secretory granules at the ultrastructural level was demonstrated. This case represented the first use of protein A-gold immunolabeling at the electron microscopic level for diagnostic purposes at our institution and exemplifies the value of this technique when the need for diagnostic immunoelectron microscopy is not anticipated. Because this tumor arose in an unusual location, ultrastructural study, including immunoelectron microscopy, not only confirmed the light microscopic diagnosis of pituitary adenoma, but further allowed subclassification of the tumor.

Aged↗

PubMed1998

Evidence using immunoelectron microscopy for regulated and constitutive pathways in the transport and release of endothelin.

We investigated the distribution of endothelin (ET)-like immunoreactivity in the human coronary artery and examined sites linked to the storage and release of intracellular proteins. Intense ET-like immunoreactivity was observed at the light-microscope level in luminal coronary artery endothelial cells. A low level of staining also was detected in the outer medial smooth-muscle layer and diffusely within the adventitia. Immunoelectron microscopy was used to determine the ultrastructural localisation of ET in the endothelium. Positive ET-like immunoreactive staining was detected in secretory vesicles at the ultrastructural level. Quantitative immunoelectron microscopy revealed that ET-like immunoreactivity was predominantly localised to the cytoplasmic matrix (including secretory vesicles) and Weibel-Palade bodies (endothelial cell-specific storage granules). Labelling was detected in 44% of Weibel-Palade body profiles positively identified by using antisera to von Willebrand factor and in cytoplasm surrounding these structures. A low level of immunoreactive staining was associated with mitochondria, whereas the cell nucleus and Golgi complex showed little or no positive staining. These findings indicate that ET is released from human coronary artery endothelial cells via two distinct secretory pathways. We propose that ET is continuously transported in and released from secretory vesicles by the constitutive secretory pathway. ET may also be stored in Weibel-Palade bodies and released after an appropriate stimulus by the regulated pathway. Positive immunoreactivity was also observed in plasmalemmal vesicles (50- to 60-nm diameter), indicating a role for these structures in endocytosis.

Adult↗

PubMed2003

Time course of Gag protein assembly in HIV-1-infected cells: a study by immunoelectron microscopy.

Recent biochemical studies have identified high molecular complexes of the HIV Gag precursor in the cytosol of infected cells. Using immunoelectron microscopy we studied the time course of the synthesis and assembly of a HIV Gag precursor protein (pr55gag) in Sf9 cells infected with recombinant baculovirus expressing the HIV gag gene. We also immunolabeled for pr55gag human T4 cells acutely or chronically infected with HIV-1. In Sf9 cells, the time course study showed that the first Gag protein appeared in the cytoplasm at 28-30 h p.i. and that budding started 6-8 h later. Colloidal gold particles, used to visualize the Gag protein, were first scattered randomly throughout the cytoplasm, but soon clusters representing 100 to 1000 copies of pr55gag were also observed. By contrast, in cells with budding or released virus-like particles the cytoplasm was virtually free of gold particles while the released virus-like particles were heavily labeled. Statistical analysis showed that between 80 and 90% of the gold particles in the cytoplasm were seen as singles, as doublets, or in small groups of up to five particles probably representing small oligomers. Clusters of gold particles were also observed in acutely infected lymphocytes as well as in multinuclear cells of chronically infected cultures of T4 cells. In a few cases small aggregates of gold particles were found in the nuclei of T4 lymphocytes. These observations suggest that the Gag polyprotein forms small oligomers in the cytoplasm of expressing cells but that assembly into multimeric complexes takes place predominantly at the plasma membrane. Large accumulations of Gag protein in the cytoplasm may represent misfolded molecules destined for degradation.

Animals↗

PubMed1999

Direct evidence for the intracellular localization of Hsp104 in Saccharomyces cerevisiae by immunoelectron microscopy.

To reveal the intracellular localization of Hsp104 in the yeast Saccharomyces cerevisiae before and after heat-shock, we performed immunoelectron microscopy after immunogold labeling with anti-Hsp104 antibody. At normal temperature (25 degrees C), a small amount of Hsp104 was located in the cytoplasm and nucleus. On exposure to mild heat-shock at 40 degrees C, protein aggregates appeared in the cytoplasm and nucleus, and Hsp104 increased around the aggregates with increasing time of the mild heat-shock treatment. Moreover, at lethal heat-shock temperature (51 degrees C) for 20 min after mild heat treatment at 40 degrees C, the intracellular localization of Hsp104 and intracellular structures were similar to those of the mild heat-shocked cells. However, in the lethally heat-shocked cells, certain intracellular structures were destroyed, and Hsp104 was not expressed. In the hsp104 null mutant strain Deltahsp104 which was treated at 40 degrees C, Hsp104 was not localized around the aggregates. Additionally, in the Deltahsp104 strain, even mild heat-shocked cells at 37 degrees C or 40 degrees C, showed destruction of intracellular structure compared to the wild-type strain. Our data suggest the following: (1) Hsp104 is associated closely with protein aggregates during heat-shock treatment, (2) Hsp104 is important for maintenance of the intracellular structure under lethal heat-shock conditions, (3) acquisition of thermotolerance depends on the amount of Hsp104 produced during mild heat-shock treatment.

Cell Nucleus↗