scholarly journals Ultrastructural localization of nuclei acids by the use of enzyme-gold complexes.

1981 ◽  
Vol 29 (4) ◽  
pp. 531-541 ◽  
Author(s):  
M Bendayan
Keyword(s):  
Secretory Granules ◽  
Ultrastructural Localization ◽  
Rnase A ◽  
Gold Complex ◽  
Gold Complexes ◽  
Thin Sections ◽  
Gold Particles ◽  
Enzyme Substrate ◽  
Cellular Compartments ◽  
Cytochemical Technique

A cytochemical technique for the ultrastructural localization of substrates using enzyme-gold complexes is reported. RNase A and DNase I have been labeled with gold particles. The RNase-gold and dNase-gold complexes obtained were applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Different cellular compartments were labeled by these enzyme-gold complexes. Using the RNase-gold complex the rough endoplasmic reticulum appeared decorated with gold particles. The gold marker was also present over the nucleus, especially over the nucleolus; mitochondria were weakly labeled. Using the DNase-gold complex, gold particles were concentrated over the euchromatin of the nucleus and the mitochondria. The heterochromatin and the nucleolus showed a less intense labeling. For both enzyme-gold complexes, the Golgi area, the secretory granules and the extracellular space appeared free of label. In those control conditions where the substrates were added to the enzyme-gold complexes a major reduction in the labeling was observed. A quantitative evaluation of the labeling was performed. This evaluation confirmed the qualitative observations and the marked reduction of labeling occurring under the control conditions. The combination of the specificity of the enzyme-substrate interactions with the size and electron density of the gold particles and the good ultrastructural preservation of the tissues resulted in a very specific labeling with high resolution. These results demonstrate the possibility of detecting substrates by means of enzyme-gold complexes at the electron microscope level.

scholarly journals High-resolution cytochemistry of neuraminic and hexuronic acid-containing macromolecules applying the enzyme-gold approach.

1988 ◽  
Vol 36 (8) ◽  
pp. 1005-1014 ◽  
Author(s):  
I Londoño ◽  
M Bendayan
Keyword(s):  
Golgi Apparatus ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Goblet Cells ◽  
Ultrastructural Level ◽  
Gold Complex ◽  
Thin Sections ◽  
Gold Particles ◽  
Tissue Sections ◽  
Apical Side

We localized acidic glycoconjugates at the ultrastructural level by applying the enzyme-gold approach. Neuraminidase and hyaluronidase were adsorbed to colloidal gold particles and applied to tissue sections under optimal conditions for their enzymatic activity. Neuraminidase-gold labeling was distributed over the Golgi apparatus and associated secretory granules in exocrine pancreatic cells and duodenal goblet cells. Mitochondria were labeled over inner membranes. Labeling was also found over the dispersed chromatin in the nucleus. Plasma membranes, particularly the apical side, were labeled by gold particles. On the other hand, incubation of tissue sections with the hyaluronidase-gold complex resulted in intense labeling of the rER membranes, the plasma membrane, and the dense chromatin in the nucleus. Labeling was also found over the Golgi apparatus and associated secretory granules, but only in duodenal goblet cells. Specificity of the results was confirmed by various control experiments performed, indicating that the enzyme-gold technique is useful for detecting linked-sugar residues on tissue thin sections. Labelings found over intra- and extracellular compartments in the present work are discussed in light of previous biochemical indications as well as of other histochemical detections of these glycoconjugates.

scholarly journals Application of lectin--gold complexes for electron microscopic localization of glycoconjugates on thin sections.

1983 ◽  
Vol 31 (8) ◽  
pp. 987-999 ◽  
Author(s):  
J Roth
Keyword(s):  
Endoplasmic Reticulum ◽  
Concanavalin A ◽  
Binding Sites ◽  
Light And Electron Microscopy ◽  
Gold Complex ◽  
Electron Microscopic ◽  
Gold Complexes ◽  
Thin Sections ◽  
Renal Tubular Cells ◽  
Lowicryl K4m

A method is described for the electron microscopic detection of lectin-binding sites in different cellular compartments and extracellular structures that uses thin sections from resin-embedded tissues. Various lectins (Ricinus communis lectin I and II, peanut lectin, Lotus tetragonolobus lectin, Ulex europeus lectin I, Lens culinaris lectin, Helix pomatia lectin, and soybean lectin) were bound to particles of colloidal gold and used for direct staining of thin sections or glycoprotein--gold complexes were prepared and applied in an indirect technique (concanavalin A and horseradish peroxidase--gold complex; wheat germ lectin and ovomucoid--gold complex). The details for preparation of such complexes from 14 nm gold particles are reported. The conditions of tissue processing that gave satisfactory staining results and good fine structure preservation were mild aldehyde fixation without osmification and low temperature embedding with the hydrophilic resin Lowicryl K4M. None of the so-called etching procedures was necessary prior to labeling of Lowicryl K4M thin sections. Examples of the use of this approach for detection of glycoconjugates in the rough endoplasmic reticulum, Golgi apparatus, and mucin of intestinal goblet cells as well as plasma membrane and various intracellular structures of absorptive intestinal and renal tubular cells are shown. A comparison is made with preembedding staining results on Concanavalin A-binding site localization in rat liver which shows that problems of penetration common in such a technique are circumvented by the postembedding approach described here. Concanavalin A-binding sites were not only consistently found in nuclear envelope, rough and smooth endoplasmic reticulum, plasma membranes, and collagen fibers, but also in mitochondria, glycogen, ribosomes, and nucleus. These data and those of a previous investigation (Roth J, Cytochem 31:547, 1983) prove the applicability of this cytochemical technique for postembedding localization of glycoconjugates by light and electron microscopy.

scholarly journals Ultrastructural localization of acidic glycoconjugates with the low iron diamine method.

1982 ◽  
Vol 30 (5) ◽  
pp. 471-476 ◽  
Author(s):  
M Takagi ◽  
R T Parmley ◽  
S S Spicer ◽  
F R Denys ◽  
M E Setser
Keyword(s):  
Bone Marrow ◽  
External Surface ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Electron Microscope Level ◽  
Thin Sections

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.

scholarly journals An immunocytochemical study on co-localization of cathepsin B and atrial natriuretic peptides in secretory granules of atrial myoendocrine cells of rat heart.

1989 ◽  
Vol 37 (3) ◽  
pp. 347-351 ◽  
Author(s):  
T Watanabe ◽  
M Watanabe ◽  
Y Ishii ◽  
H Matsuba ◽  
S Kimura ◽  
...  
Keyword(s):  
Natriuretic Peptides ◽  
Cathepsin B ◽  
Rat Heart ◽  
Secretory Granules ◽  
Atrial Natriuretic Peptides ◽  
Thin Sections ◽  
Gold Particles ◽  
Atrial Tissue ◽  
Myoendocrine Cells ◽  
Atrial Natriuretic

To examine localization of cathepsin B, a representative lysosomal cysteine protease, in atrial myoendocrine cells of the rat heart, immunohistochemistry at the light and electron microscopic level was applied to the atrial tissue, using a monospecific antibody for rat liver cathepsin B. In serial semi-thin sections, immunoreactivity for cathepsin B and atrial natriuretic peptides (ANP) was detected in the para-nuclear region of atrial myoendocrine cells. Several large granules and many fine granules in the region of the cells were positively stained by the cathepsin B antibody. Gold particles indicating cathepsin B antigenicity labeled secretory granules in the cells, which were also labeled by those indicating ANP, using thin sections of the Lowicryl K4M-embedded material. Moreover, some granules labeled densely by immunogold particles for cathepsin B seemed to be lysosomes. By double immunostaining using thin sections of the Epon-embedded material, gold particles indicating cathepsin B and ANP antigenicities were co-localized in secretory granules of the cells. By enzyme assay, activity of cathepsin B was three times higher in atrial tissue than ventricular tissue. The results suggest that co-localization of cathepsin B and ANP in secretory granules is compatible with the possibility that cathepsin B participates in the maturation process of ANP.

scholarly journals Ultrastructural localization of glucoside residues on tissue sections by applying the enzyme-gold approach.

1987 ◽  
Vol 35 (10) ◽  
pp. 1149-1155 ◽  
Author(s):  
M Bendayan ◽  
N Benhamou
Keyword(s):  
Secretory Granules ◽  
Ultrastructural Localization ◽  
Wall Material ◽  
Gold Complex ◽  
Plant Tissues ◽  
Fungal Cell ◽  
Tissue Sections ◽  
Pancreatic Cells ◽  
Beta Glucosidase ◽  
Labeling Pattern

The enzyme-gold approach was applied for ultrastructural localization of glucoside residues in animal and plant tissues. A beta-glucosidase-gold complex was prepared and used on thin tissue sections to reveal the corresponding substrate molecules by electron microscopy. Conditions for preparation of the complex, as well as for its application, were determined. Once applied on thin tissue sections, the glucosidase-gold complex yielded labeling over the rough endoplasmic reticulum, mainly on the ribosomal side of the membranes, and over the dense chromatin in the nucleus. Mitochondria, Golgi apparatus, and secretory granules in liver and pancreatic cells were free of gold particles. In plant cells, the labeling pattern was similar. In addition, the stroma regions of chloroplasts were densely labeled. In the extracellular space, labeling was found over the basal laminae of cells in animal tissues and over the fibrillar wall material bordering the intercellular space in plant tissues. Fungal cell cytoplasm was also labeled, as well as the membrane delineating mycoplasma-like organisms. Control conditions confirmed these labelings, demonstrating the possibility of revealing glucoside residues on tissue sections with high resolution and specificity.

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 Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique.

1983 ◽  
Vol 31 (1) ◽  
pp. 101-109 ◽  
Author(s):  
M Bendayan ◽  
M Zollinger
Keyword(s):  
Secretory Granules ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Liver Mitochondria ◽  
Secretory Proteins ◽  
Additional Advantage ◽  
Sodium Metaperiodate ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Structural Preservation

The protein A-gold immunocytochemical technique has been modified to allow labeling of cellular antigenic sites on osmium-fixed or postfixed tissues. Several strong oxidizing agents have been found able to restore protein antigenicity on osmicated tissue thin sections. According to the fine structural preservation and intensities of labeling, pretreatment with sodium metaperiodate gave optimal results. Pancreatic secretory proteins (and/or proproteins) as well as insulin (and/or proinsulin) were localized over perfectly preserved rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules of the corresponding pancreatic cells; carbamyl phosphate synthetase and catalase were revealed over liver mitochondria and peroxisomes, respectively. In addition to the higher resolution in the labeling obtained using osmium-fixed tissues, the present modification confers an additional advantage to the protein A-gold technique by allowing labeling on tissues processed for routine electron microscopy.

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