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At least 145 records · Page 8Linked to original sources
PubMed1992

Immunoelectron microscopy of carbonic anhydrase isozyme VI in rat submandibular gland: comparison with isozymes I and II.

Carbonic anhydrase (CA) was purified from the saliva of pilocarpine-treated rats by inhibitor-affinity chromatography, and its localization in the rat submandibular gland was studied by the indirect immunoperoxidase technique using a monoclonal antibody (MAb) raised against the enzyme. SDS-polyacrylamide gel electrophoresis of the CA VI gave three bands of 33, 39, and 42 KD. Enzyme digestion experiment showed that the 42 KD molecule was degraded into the 39 KD molecule and the 39 KD molecule into the 33 KD molecule. The cleavage of the 42 KD molecule was independent and that of the 39 KD molecule was dependent on endo-beta-N-acetylglucosaminidase F. The 42 KD molecule was detected in the CA purified from the pilocarpine-treated but not the untreated salivary gland. The MAb recognized all the three components of the enzyme. Immunostaining for CA VI was seen in the cytosol and secretory granules of serous acinar cells and in the duct luminal contents. Staining specific for erythrocyte CA (CA I and CA II) was observed in the cytosol of the epithelial cells of granular, striated, and excretory ducts. Among these duct cells, the agranular varieties in the granular and excretory ducts were essentially devoid of the immunoreactivity.

Animals↗
PubMed1993

Distribution of undulin, tenascin, and fibronectin in the human periodontal ligament and cementum: comparative immunoelectron microscopy with ultra-thin cryosections.

We studied the ultrastructural localization of three distantly related glycoproteins of the extracellular matrix, undulin, tenascin and fibronectin, in decalcified sections of human periodontal ligament (PL) and cementum. Undulin was associated with tightly packed major collagen fibrils and not with microfibrils, indicating that this protein may be involved in the supramolecular and functional organization of collagen fibrils into flexible bundles. Tenascin was found on globular masses between less densely packed collagen fibrils, thus displaying a pattern quite distinct from that of undulin. Fibronectin was noted in bulky material between the cross-striated fibrils, often surrounding individual fibrils like garlands, and in the microfibrillar meshwork extending from cross-striated fibrils. The three glycoproteins displayed a distinct and unique pattern of distribution in PL that can be correlated with their molecular structure and potential functions.

Adolescent↗
PubMed1993

Immunoelectron microscopy of carbonic anhydrase isozyme VI in human submandibular gland: comparison with isozymes I and II.

Carbonic anhydrase VI (CA VI) was purified from human saliva by inhibitor-affinity chromatography, and its distribution was studied in human submandibular gland by the indirect immunoperoxidase technique with a rabbit polyclonal antibody raised against the isozyme. Polyclonal antibodies to human CA I and CA II purified from erythrocytes were also raised and used for immunostaining. SDS-polyacrylamide gel electrophoresis of the purified isozymes revealed a single protein band (CA VI, 42 KD; CA I and CA II, 30 KD). Antibody raised against CA VI did not crossreact with CA I or CA II either by Western or by dot-blotting. However, antibodies against CA I and CA II showed slight crossreaction with each other's antigen by dot-blotting. In a Western blot of purified submandibular gland CA, antibody to CA VI stained the 42 and 30 KD bands, and antibodies to CA I and CA II stained the 30 KD band. The 42 KD but not the 30 KD molecule was cleaved by endo-beta-N-acetylglucosaminidase F, indicating that the former contains N-linked oligosaccharides. Immunostaining for CA VI was seen in the secretory granules and cytosol of serous acinar cells and in the duct luminal contents. Staining specific for CA II was observed in the cytosol of serous acinar and duct epithelial cells. Antibody to CA I reacted only with the walls of small blood vessels. These results suggest that (a) serous acinar cells secrete 42 KD CA VI which functions in the oral cavity and that (b) serous acinar and duct epithelial cells possess cytosolic CA (30 KD CA VI and CA II) which functions in situ.

Blotting, Western↗
PubMed1995

Antigen unmasking for immunoelectron microscopy: labeling is improved by treating with sodium ethoxide or sodium metaperiodate, then heating on retrieval medium.

To optimize the ultrastructural localization of immunoglobulin G in corneal crystalloid deposits, we compared a range of antigen unmasking techniques. A human corneal biopsy specimen was fixed in formalin, post-osmicated, and embedded in epoxy resin for electron microscopy. Thin sections were immunogold-labeled for IgG after treatment with sodium ethoxide or sodium metaperiodate. Sections were also treated by heating them at 95 degrees C while they floated on water, 0.01 M citrate buffer (pH 6.0), or sodium metaperiodate. The treatments were applied separately and combined. After labeling, crystalloids in untreated sections had a probe density of 5 particles/microns2. Crystalloids in sections treated only with sodium ethoxide or sodium metaperiodate had probe densities of 15-20 particles/microns2. Sodium ethoxide combined with heating on water, or citrate buffer, gave probe densities of 140-160 particles/microns2. Sodium metaperiodate combined with heating on citrate buffer gave the highest probe density (195 particles/microns2). Although sodium ethoxide coupled with heating increased probe density, the ethoxide etched the sections and caused unacceptable damage. Treatment with sodium metaperiodate followed by heating on citrate buffer is recommended for antigen unmasking. This combination gave a high probe density and sections remained intact, with good ultrastructural detail.

Cornea↗
PubMed1990

Nucleolar distribution of proteins B23 and nucleolin in mouse preimplantation embryos as visualized by immunoelectron microscopy.

The ultrastructural distribution of proteins B23 and nucleolin in the nucleolus of mouse embryos from the zygote to the early blastocyst has been analyzed by means of specific antibodies and immunocytochemistry using colloidal gold complexes as markers. In parallel, silver staining of nucleoli was carried out on ultrathin sections. Our results show that the compact prenucleolar bodies at 1- and 2-cell stage as well as the compact residual fibrillar masses observed up to the morula stage, are labelled with the two antibodies. These masses, however, are not stained with silver up to the 4-cell stage. In well-developed nucleoli, the two antibodies co-localize in the dense fibrillar component (DFC) and the granular component (GC) while fibrillar centers (FCs) are devoid of label. On the contrary, silver staining occurs in the FCs and DFC but not in the GC. Our observations suggest that there is no direct relationship between the occurrence of silver staining and the distribution of protein B23 or nucleolin. Moreover, neither the localization of the two above proteins nor silver staining are unequivocally related to the nucleolar activity.

Animals↗
PubMed2006

The histopathology of Candida albicans invasion in neonatal rat tissues and in the human blood-brain barrier in culture revealed by light, scanning, transmission and immunoelectron microscopy.

The present studies examined the effects of Candida albicans yeast and hyphal morphologies on tissue pathologies and transmigration properties of the fungus in two experimental models: 1) an in vivo, neonatal rat model, and 2) a cell culture model of human brain microvascular endothelial cells (ECs) (BMVEC). We inoculated a hyphae-producing strain (CAI4-URA3) and a non-hyphae-producing strain (CAI4) of C. albicans into 4-10 day old rats and BMVEC cultures. Animals were inoculated by intraperitonal (i.p.), intranasal (i.n.), oral (p.o.) and intracerebral (i.c.) routes and several tissues were examined after 24-48 hrs. Rats inoculated i.p. with the hyphae-producing strain showed pathology in the kidneys, liver, spleen, and other tissues associated with inoculation tracks of the nose, and muscle and connective tissues of the abdominal wall. Few animals inoculated i.p., however, presented evidence of meningitis. The non-hyphae phase yeast produced neither tissue pathology nor meningitis. Animals inoculated i.c. with the hyphae strain after 1 and 3 hrs expressed minimal meningitis, with an increasing neutrophillic meningitis between 4 and 18 hrs after inoculation. At 18 hrs after i.c. inoculation, however, the inflammatory foci and brain pathology were extensive and demonstrated mycelia within the lateral ventricles associated with necrosis of adjacent brain tissue. Neutrophillic meningitis at this time period was pronounced. BMVEC co-cultured 1-2 hrs with both C. albicans strains showed EC phagocytosis of hyphae and blastospores into intercellular adhesion molecule-1 (ICAM-1)-labeled caveolae suggesting a transcellular role for ICAM-1 in the internalization process of C. albicans.

Animals↗
PubMed1993

The localization of synaptophysin in the organ of Corti of the human as shown by immunoelectron microscopy.

Synaptophysin, or p38, a polypeptide of molecular weight 38 kD, is a calcium-binding membrane protein found in synaptic vesicles of neurons and smooth surfaced vesicles of neuroendocrine cells. Six human neonatal and infant temporal bones were fixed in paraformaldehyde and glutaraldehyde, decalcified in EDTA and were than immunoreacted for synaptophysin (ICN Biomedicals) using the avidin-biotin reaction (ABC kit, Vector Labs). The tissue was then prepared for light microscopic surface preparation, radial sections of 5 microns, and serial section electron microscopy. At a light microscopic level, the inner spiral bundle, tunnel spiral bundle, upper tunnel crossing fibers and the base of outer hair cells were stained. At the base of outer hair cells, the immunoreactivity was seen to decrease from the base to the apex and from the first to third outer hair cells. At an electron microscopic level, immunoreactivity at the base of outer hair cells was limited to vesiculated efferent fibers. The degree of immunoreactivity between adjacent efferent fibers varied significantly. Immunoreactive vesiculated endings were also found in the supranuclear region of outer hair cells.

Hair Cells, Auditory↗
PubMed1991

Differential silver enhanced double labeling in immunoelectron microscopy.

A two-sided double labeling method using protein A gold was used to demonstrate the presence of two hormones within the same anterior pituitary cell granule. A single probe size was used for both section faces but one side of the grid was silver enhanced. The use of a single probe size reduced the cost of the study and eliminated the variations in labeling efficiency that result from the use of different probe sizes.

Follicle Stimulating Hormone↗
PubMed1996

A new fixation procedure for study of the histaminergic neurons by immunoelectron microscopy using the direct antiserum against histamine.

A new perfusion protocol was developed to detect histamine-like immunoreactive neurons at the electron microscopic level. By stepwise perfusion of 1-ethyl-3(3-diamethylaminopropyl)-carbodiimide and paraformaldehyde solutions, the brain block could be cut with a vibratome and the immunoreactivity could be detected using the avidin-biotin-peroxidase-complex method. We used this method to study the ultrastructure and synaptic relations of the histaminergic neurons in the postmammillary caudal magnocellular nucleus of the rat hypothalamus. This method should also be useful for examination of histaminergic neurons in other tissues and the synaptic relations of histaminergic neurons with other neurotransmitter-containing neurons by double immunostaining.

Animals↗
PubMed1998

Recognition of Pneumocystis carinii antigen on its surface by immunohistochemistry and immunoelectron microscopy.

The aim of this study was to detect the surface antigens in different stages of experimental induced Pneumocystis carinii in Sprague-Dawley rats. Immunohistochemical staining with monoclonal (900, 902 and 904) and polyclonal (SP-D) antibodies demonstrated that the P. carinii organisms were mostly in the alveolar lumina. The binding sites of the monoclonal (900, 902 and 904) and polyclonal (SP-D) antibodies developed against P. carinii were examined at the ultrastructural level by using a post-embedding immunogold labeling. The gold particles were observed evenly on the surface of precyst and cyst stages of the P. carinii. In the trophozoite stage, scattered gold particles were seen on the pellicles and tubular expansions. The monoclonal antibodies reacted mainly with pellicles of P. carinii, whereas SP-D labeled pellicles, intracystic bodies, cytoplasms of alveolar macrophages, free floating surfactant material in the alveolar spaces, and adjacent type II epithelial cells. In the immunogold labeling, basically no significant differences were found in the precyst, cyst, and ruptured cyst stages. These results indicate that the gold particles were observed adhering to every stage of P. carinii, mostly concentrated on the pellicles, and more concentrated in the precyst or cyst stage than trophozoite stage which may be due to an increase in antigen accumulation during development from the trophozoite to the cyst.

Animals↗
PubMed2001

[Localization of immunodominant antigens of the liver fluke Opisthorchis felineus by immunoelectron microscopy].

Immune electron microscopy with a panel of monoclonal antibodies to O. felineus antigens and human immune sera from patients was used for localization of the main antigens of adult O. felineus. The immune complexes at the ultrastructural level were visualized by 5-nm colloidal gold. The main antigens recognized by human antibodies and monoclonal antibodies were associated with the tegument, muscles, uterus, gonads, intestine and eggs of the liver fluke. The findings led to the conclusion that the surface structures of liver flukes stimulate a low B-cell immune response. The structures of the excretory-secretory system of the parasite and their products contain a lot of main antigens and induce B-immune response in man.

Animals↗
PubMed2002

Distribution of actin in etoposide-induced human leukemia cell line K-562 using fluorescence and immunoelectron microscopy technique.

Localization of actin was studied in erythroleukemic cell line K-562 after treatment with etoposide for 72 hours in a range of concentrations 0.02-200 microM/L. Actin was visualised by fluorescence microscopy and streptavidingold method. These findings indicate that changes in actin after treatment with etoposide were dose-dependent. Significant changes in the cellular distribution of F-actin in K-562 cells were obtained after treatment with 20 and 200 microM/L etoposide. In comparison with control cells, the number of the cells decreased and cells were larger especially at 200 microM/L. F-actin was diffusely distributed throughout the cell at 20 microM/L. Treatment of cells with 200 microM/L etoposide showed F-actin diffusely distributed throughout the cell with local actin assemblies and also at the cell periphery. Immunogold labelling of actin was observed in cells treated with all doses of etoposide and control cells. Labelling was found in the nucleus and also in the cytoplasm. At the ultrastructural level, cells treated with 200 microM/L etoposide showed protrusions at the surface, in which increase of actin was often observed. Etoposide causes changes in actin distribution of K-562 cells, and the changes in expression of actin were not only restricted to cell with features of apoptosis.

Actins↗
PubMed1992

Dynamic redistribution of major platelet surface receptors after contact-induced platelet activation and spreading. An immunoelectron microscopy study.

The authors used an immunogold labeling procedure to investigate the redistribution of platelet receptors and their ligands on the surface of contact-activated adherent platelets before and after thrombin stimulation. During the initial stage of platelet adhesion, a typical segregation of receptors occurred. Gold particles identifying glycoprotein (GP) Ib (CD42b) and GPIIb-IIIa (CD41a) remained distributed over the entire platelet surface, whereas gold particles identifying GPIa-IIa (CDw 49b) and GPIV (CD36) were found essentially overlying the granulomere; p24 (CD9) was present at the peripheral platelet rim and over the cell body. An increased labeling of GPIIb-IIIa, GPIV and p24 was also observed on pseudopods, with GPIIb-IIIa and GPIV concentrated at the enlarged extremities and at sites of contact between two platelets, whereas GPIb was absent from pseudopods. After thrombin stimulation of adherent platelets, GPIb underwent a relocation to the cell center, in contrast to GPIIb-IIIa which still remained randomly distributed over the cell body. To investigate whether ligand distribution paralleled this receptor segregation, platelet released von Willebrand factor (vWF), fibrinogen (Fg) and thrombospondin (TSP) were visualized. During the early stages of platelet activation, surface labeling for all three adhesive proteins was minimal and almost undetectable. Occasionally, intragranular Fg and vWF was accessible to gold-coupled antibodies, with vWF exhibiting the typical eccentric alpha-granular localization. At later stages of activation and especially after thrombin stimulation, no surface labeling for vWF was observed, whereas immunogold particles identifying vWF were still present inside enlarged clear vacuoles. In contrast, labeling of Fg and TSP was increased over the granulomere and extended to the cell periphery and the pseudopods, but was absent from the hyalomere, despite the presence of GPIIb-IIIa molecules. Double labeling experiments showed colocalization of Fg and TSP, GPIV and TSP, as well as Fg and GPIIb-IIIa, although no typical coclustering of GPIIb-IIIa and GPIV or GPIIb-IIIa and p24 was apparent. Our results further suggest that 1) on surface activated adherent platelets, not all GPIIb-IIIa molecules become competent to bind Fg, 2) GPIa-IIa is not anchored to the platelet membrane skeleton, and 3) during the early stage of platelet activation, a communication exists between the alpha granules and the platelet surface.

Antigens, CD↗