scholarly journals The detection of substructures within proteoglycan molecules. Electron-microscopic immuno-localization with the use of Protein A-gold

1987 ◽  
Vol 247 (2) ◽  
pp. 267-276 ◽  
Author(s):  
J K Sheehan ◽  
A Ratcliffe ◽  
K Oates ◽  
T E Hardingham
Keyword(s):  
Monoclonal Antibody ◽  
Chondroitin Sulphate ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Electron Microscopic ◽  
Keratan Sulphate ◽  
Gold Particles ◽  
Protein Core ◽  
Gold Labelling ◽  
Chondroitin Ac Lyase

Proteoglycan monomers from pig laryngeal cartilage were examined by electron microscopy with benzyldimethylalkylammonium chloride as the spreading agent. The proteoglycans appeared as extended molecules with a beaded structure, representing the chondroitin sulphate chains collapsed around the protein core. Often a fine filamentous tail was present at one end. Substructures within proteoglycan molecules were localized by incubation with specific antibodies followed by Protein A-gold (diameter 4 nm). After the use of an anti-(binding region) serum the Protein A-gold (typically one to three particles) bound at the extreme end of the filamentous region. A small proportion of the labelled molecules (10-15%) showed the presence of gold particles at both ends. A monoclonal antibody specific for a keratan sulphate epitope (MZ15) localized a keratan sulphate-rich region at one end of the proteoglycan, but gold particles were not observed along the extended part of the protein core. This distribution was not changed by prior chondroitin AC lyase digestion of the proteoglycan. Localization with a different monoclonal antibody to keratan sulphate (5-D-4) caused a change in the spreading behaviour of a proportion (approx. 20%) of the proteoglycan monomers that lost their beaded structure and appeared with the chondroitin sulphate chains projecting from the protein core. In these molecules the Protein A-gold localized antibody (5-D-4) along the length of the protein core whereas in those molecules with a beaded appearance it labelled only at one end. Labelling with either of the monoclonal antibodies was specific, as it was inhibited by exogenously added keratan sulphate. The differential localization achieved may reflect structural differences within the proteoglycan population involving keratan sulphate and the protein core to which it is attached. The results showed that by this technique substructures within proteoglycan molecules can be identified by Protein A-gold labelling after the use of specific monoclonal or polyclonal antibodies.

scholarly journals Electron microscopic localization of glutamate dehydrogenase in rat liver mitochondria by an immunogold procedure and monoclonal and polyclonal antibodies.

1986 ◽  
Vol 34 (7) ◽  
pp. 913-922 ◽  
Author(s):  
E Knecht ◽  
A Martinez-Ramon ◽  
S Grisolia
Keyword(s):  
Glutamate Dehydrogenase ◽  
Rat Liver ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Electron Microscopic ◽  
Cross Sectional ◽  
Gold Particles ◽  
Parenchymal Cells

Glutamate dehydrogenase (GDH) was localized in rat liver by indirect electron microscopic immunogold, using different sizes of gold particles and monoclonal and polyclonal antibodies. Using the protein A-gold technique in double immunocytochemical experiments, both antibodies, at their optimal dilutions, gave similar results. A novel assessment of the distribution of GDH was made by measurements of the number of gold particles per square micrometer of cross-sectional images of individual mitochondria. The data indicate intracellular homogeneity among mitochondria in individual parenchymal cells. The enzyme is almost absent in non-parenchymal cells. Finally, GDH was found mainly in association with the mitochondrial inner membrane.

scholarly journals Immunoelectron microscopic localization of an oviductal antigen in hamster zona pellucida by use of a monoclonal antibody.

1988 ◽  
Vol 36 (11) ◽  
pp. 1441-1447 ◽  
Author(s):  
F W Kan ◽  
S St-Jacques ◽  
G Bleau
Keyword(s):  
Monoclonal Antibody ◽  
Zona Pellucida ◽  
Preimplantation Embryo ◽  
Protein A ◽  
Cumulus Cells ◽  
Gold Particles ◽  
Antigenic Sites ◽  
Cumulus Oophorus ◽  
Entire Thickness ◽  
Immunocytochemical Method

The zona pellucida is an extracellular matrix of glycoproteins which surrounds the mammalian oocyte and preimplantation embryo. We have recently developed monoclonal antibodies against oviductal zona pellucida of the golden hamster. We applied the post-embedding immunocytochemical method using a monoclonal antibody (IgGl,k) to determine the precise location of antigenic sites in the cumulus oophorus complex of the superovulated hamster. By applying the high-resolution protein A-gold technique, we demonstrated that the sites of immunoreactivity were exclusively in the zona pellucida encompassing the oocyte. Other structures within the oocyte and neighboring cumulus cells were not labeled by gold particles. Moreover, gold particles were evenly distributed throughout the entire thickness of the zona pellucida, indicating that this extracellular layer is at least in part made up of an antigen recognized by the monoclonal antibody that is uniformly distributed in the zona matrix.

scholarly journals Immunocytochemical localization of D-amino acid oxidase in the central clear matrix of rat kidney peroxisomes.

1986 ◽  
Vol 34 (12) ◽  
pp. 1709-1718 ◽  
Author(s):  
N Usuda ◽  
S Yokota ◽  
T Hashimoto ◽  
T Nagata
Keyword(s):  
Amino Acid ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Protein A ◽  
Immunoblot Analysis ◽  
Acid Oxidase ◽  
Electron Microscopic ◽  
Amino Acid Oxidase ◽  
Thin Sections ◽  
Gold Particles

Light and electron microscopic localizations of D-amino acid oxidase (DAO) in rat kidney was investigated using immunoenzyme and protein A-gold techniques. The enzyme was purified from rat kidney homogenate and its antibody was raised in rabbits. By Ouchterlony double-diffusion analysis and immunoblot analysis with anti-(rat kidney DAO) immunoglobulin, the antibody was confirmed to be monospecific. The tissue sections (200 micron thick) of fixed rat kidney were embedded in Epon or Lowicryl K4M. Semi-thin sections were stained for DAO by the immunoenzyme technique after removal of epoxy resin for LM, and ultra-thin sections of Lowicryl-embedded material were labeled for DAO by the protein A-gold technique for EM. By LM, fine cytoplasmic granules of proximal tubule were stained exclusively. Among three segments of proximal tubules, and S2 and S3 segments were heavily stained but the S1 segment only weakly so. By EM, gold particles indicating the antigenic sites for DAO were exclusively confined to peroxisomes. Within peroxisomes, the gold particles were localized in the central clear matrix but not in the peripheral tubular substructures. The results indicate that D-amino acid oxidase in rat kidney is present exclusively in peroxisomes in the proximal tubule and that within peroxisomes it is found only in central clear matrix and not in the peripheral tubular substructures.

scholarly journals Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles.

1986 ◽  
Vol 103 (6) ◽  
pp. 2273-2281 ◽  
Author(s):  
L Orci ◽  
M Ravazzola ◽  
M Amherdt ◽  
O Madsen ◽  
A Perrelet ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Islet Cells ◽  
Protein A ◽  
Secretory Vesicle ◽  
Average Density ◽  
Secretory Vesicles ◽  
Gold Particles ◽  
Single Polypeptide Chain ◽  
Igg Binding ◽  
Single Polypeptide

Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H+ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4-dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation. The cells were processed for indirect protein A-gold colocalization of DAMP, using a monoclonal antibody to dinitrophenol, and proinsulin, using a monoclonal antibody that exclusively reacts with the prohormone. The average density of DAMP-specific gold particles in immature secretory vesicles that contained proinsulin was 71/micron 2 (18 times cytoplasmic background), which indicated that this compartment was acidic. However, the density of DAMP-specific gold particles in the insulin-rich mature secretory vesicle averaged 433/micron 2. This suggests that although proinsulin conversion occurs in an acidic compartment, the secretory vesicles become more acidic as they mature. Since the concentration of anti-proinsulin IgG binding in secretory vesicles is inversely proportional to the conversion of proinsulin to insulin, we were able to determine that maturing secretory vesicles had to reach a critical pH before proinsulin conversion occurred.

scholarly journals Proteoglycans of the intervertebral disc. Homology of structure with laryngeal proteoglycans

1979 ◽  
Vol 179 (3) ◽  
pp. 561-572 ◽  
Author(s):  
R L Stevens ◽  
R J Ewins ◽  
P A Revell ◽  
H Muir
Keyword(s):  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Structural Model ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Keratan Sulphate ◽  
Protein Core ◽  
Uronic Acid Content ◽  
Normal Human ◽  
Cartilage Proteoglycans

The structure of the proteoglycans from normal pig nucleus pulposus and relatively normal human annulus fibrosus and nucleus pulposus was investigated in detail and the results were compared with the current structural model of proteoglycans of hyaline cartilage. Like proteoglycans of cartilage, those of intervertebral disc contain keratan sulphate and chondroitin sulphate attached to a protein core; they are able to aggregate to hyaluronic acid; the protein core likewise has three regions, one lacking glycosaminoglycans, another rich in keratan sulphate and a third region rich in chondroitin sulphate. However, disc proteoglycans contain more keratan sulphate and protein and less chondroitin sulphate and are also considerably smaller than cartilage proteoglycans. In proteoglycans of human discs, these differences appeared to be due principally to a shorter region of the core protein bearing the chondroitin sulphate chains, whereas in proteoglycans of pig discs their smaller size and relatively low uronic acid content were due to shorter chondroitin sulphate chains. There were subtle differences between proteoglycans from the nucleus and annulus of human discs. In the latter a higher proportion of proteoglycans was capable of binding to hyaluronate.

scholarly journals Freeze-drying technique in electron microscopic immunohistochemistry.

1985 ◽  
Vol 33 (5) ◽  
pp. 485-490 ◽  
Author(s):  
S Hisano ◽  
T Adachi ◽  
S Daikoku
Keyword(s):  
Freeze Drying ◽  
Secretory Granules ◽  
Protein A ◽  
Electron Microscopic ◽  
Gold Particles ◽  
Axon Terminals ◽  
Conventional Procedure ◽  
Small Gold Particle ◽  
Excellent Preservation ◽  
Gold Label

Postembedding immunocytochemical labeling was performed on sections of rat neurohypophysis prepared by either freeze-drying, vapor fixation and Spurr resin embedding, or conventional aqueous fixation and Spurr resin embedding. Arginine vasopressin (AVP) and oxytocin (OXT) were immunolabeled with protein A-gold-anti-AVP and protein A-gold-anti-OXT complexes, respectively. The freeze-drying procedure (FD) resulted in excellent preservation of ultrastructure and greater antigenicity than the conventional procedure (Con). More gold particles were seen over secretory granules in FD sections than in Con sections. In addition, in FD sections, the gold label was restricted to secretory granules while in Con sections, both the granules and the extragranular axoplasm exhibited label. The two antigens in FD sections could be labeled simultaneously with protein A-small gold particle-anti-OXT complex and protein A-large gold particles-anti-AVP complex. In this way the two antigens were seen to be present in secretory granules within different axon terminals. Thus FD preparations should be useful for demonstrating the presence of multiple antigens in the same granules of nerve terminals.

scholarly journals Immunocytochemical localization of cathepsin B in rat kidney. II. Electron microscopic study using the protein A-gold technique.

1986 ◽  
Vol 34 (7) ◽  
pp. 899-907 ◽  
Author(s):  
S Yokota ◽  
H Tsuji ◽  
K Kato
Keyword(s):  
Cathepsin B ◽  
Rat Kidney ◽  
Protein A ◽  
Proximal Tubules ◽  
Gold Complex ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Gold Particles ◽  
Apical Vesicles ◽  
Collecting Tubules

Thin sections of Lowicryl K4M-embedded materials were labeled with protein A-gold complex. Gold particles representing the antigen sites for cathepsin B were exclusively confined to lysosomes of each segment of the nephron. The heaviest labeling was noted in the lysosomes of the S1 segment of the proximal tubules. Labeling intensity varied considerably with the individual lysosomes. Lysosomes of the other tubular segments, such as the S2 and S3 segments of the proximal tubules, distal convoluted tubules, and collecting tubules were weakly labeled by gold particles. Quantitative analysis of labeling density also confirmed that lysosomes in the S1 segment have the highest labeling density and that approximately 65% of labeling in the whole renal segments, except for the glomerulus, was found in the S1 segment. These results indicate that in rat kidney the lysosomes of the S1 segment are a main location of cathepsin B. Further precise observations on lysosomes of the S1 segment revealed that apical vesicles, tubules, and vacuoles were devoid of gold particles, but when the vacuoles contained fine fibrillar materials, gold labeling was detectable in such vacuoles. As the lysosomal matrix becomes denser, the labeling density is increased. Some small vesicles around the Golgi complex were also labeled. These results indicate that the endocytotic apparatus including the apical vesicles, tubules, and vacuoles contains no cathepsin B. When the vacuoles develop into phagosomes, they acquire this enzyme to digest the absorbed proteins.

scholarly journals Monoclonal antibodies to different epitopes on a cell-surface enzyme, human placental alkaline phosphatase, effect different patterns of labeling with protein A-colloidal gold.

1987 ◽  
Vol 35 (11) ◽  
pp. 1277-1284 ◽  
Author(s):  
R Jemmerson ◽  
M Agre
Keyword(s):  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Colloidal Gold ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Placental Alkaline Phosphatase ◽  
Gold Particles ◽  
Human Placental Alkaline Phosphatase ◽  
Membrane Components

Two monoclonal antibodies (mAbs) to different epitopes on human placental alkaline phosphatase (PLAP), both of the immunoglobulin G2a heavy-chain class and having similar affinities for PLAP, were compared for their ability to label the enzyme on the HeLa cell surface. In one type of experiment employing [125I]-labeled mAbs, the results demonstrated quantitative differences in binding of the mAbs to the cells. At saturating levels, the number of molecules of mAb E5 bound to the cells was almost eight times the number of mAb B10 molecules bound. In another type of experiment, mAbs were indirectly visualized on the cell surface using protein A tagged with colloidal gold particles in transmission electron microscopy. Only one of the antibodies (E5) displayed a clustered distribution of PLAP that previously had been observed with rabbit polyclonal antibodies and goat anti-rabbit IgG-labeled gold (J Histochem Cytochem 33:1227, 1985). The other antibody (B10) showed less frequent and more scattered labeling; three to four times more gold particles were visualized in each cluster on cells bound by mAb E5 compared to cells bound by B10. These results are consistent with the idea that not all epitopes on a membrane-bound antigen may be equally accessible for antibody binding. Even identical epitopes on different PLAP molecules are not equally hindered by other membrane components, since at least some of the PLAP molecules are labeled by the more sterically hindered mAb B10. Quantification of the number of gold particles employing the more abundantly bound mAb E5 provides an average estimate of seven to eight molecules of PLAP in each cluster. Because of inefficiencies in labeling, however, this value is probably lower than the real number.

scholarly journals Absence of keratan sulphate from skeletal tissues of mouse and rat

1985 ◽  
Vol 228 (2) ◽  
pp. 443-450 ◽  
Author(s):  
G Venn ◽  
R M Mason
Keyword(s):  
Monoclonal Antibody ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Lumbar Disc ◽  
Heparan Sulphate ◽  
Costal Cartilage ◽  
Animal Experiments ◽  
Keratan Sulphate

The absence of keratan sulphate synthesis from skeletal tissues of young and mature mice and rats has been confirmed by (1) analysis of specific enzyme degradation products of newly synthesized glycosaminoglycans, and (2) immunohistochemistry and radioimmunoassay using a monoclonal antibody directed against keratan sulphate. Approx. 98% of the [35S]glycosaminoglycans synthesized in vivo by mouse and rat costal cartilage, and all of those of lumbar disc, are chondroitin sulphate. The remainder in costal cartilage were identified as heparan sulphate in mature rats. In contrast, [35S]glycosaminoglycans synthesized by cornea of both species comprised both chondroitin sulphate and keratan sulphate. In mice keratan sulphate accounted for 12-25% and in rats 40-50% of the total [35S]glycosaminoglycans, depending on the age of the animal. Experiments in vitro with organ culture of cartilage and cornea confirm these results. Absence of keratan sulphate from mouse costal cartilage and lumbar disc D1-proteoglycans was corroborated by inhibition radioimmunoassay with the monoclonal antibody MZ15 and by lack of staining for keratan sulphate in indirect immunofluorescence studies using the same antibody.

Fluorescent and Electron Microscopic Studies Suggest That Confronting Cisternae Arise from Nuclear Envelope in Mitotic Cells

1998 ◽  
Vol 4 (S2) ◽  
pp. 1028-1029
Author(s):  
J. R. Palisano ◽  
J. L. Thacker ◽  
C. S. Piromalli ◽  
A. M. Morrison ◽  
J. E. Tate
Keyword(s):  
Monoclonal Antibody ◽  
Nuclear Envelope ◽  
Hela Cells ◽  
Uv Light ◽  
Fluorescent Microscopy ◽  
Protein A ◽  
Specific Protein ◽  
Electron Microscopic ◽  
Lamin B ◽  
Microscopic Studies

The origin and role of confronting cisternae (CC) have been clouded in mystery since their discovery in 1955. Early investigations suggested that CC arose from either nuclear envelope (NE) or by stacking of endoplasmic reticulum (ER) cisternae. Electron microscopic studies of mitotic HeLa cells suggest that CC are fragments of NE (Fig. 1) that arise as portions of the NE fold back on one another. Because lamin B is a NE specific protein, a monoclonal antibody to lamin B was used to probe the origin of CC. The monoclonal antibody to lamin B was then detected by utilizing a secondary antibody conjugated to the fluorochrome rhodamine which fluoresces red when exposed to UV light. Fluorescent microscopy of interphase HeLa cells demonstrates that only the NE fluoresces red when the cells are probed with a monoclonal antibody to lamin B (Fig. 2). There is no label localized in the cytoplasm which supports the evidence that lamin B is only localized to the NE in interphase cells.

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