scholarly journals COMBINED CYTOCHEMICAL AND ELECTRON MICROSCOPIC DEMONSTRATION OF ß-GLUCURONIDASE ACTIVITY IN RAT LIVER WITH THE USE OF A SIMULTANEOUS COUPLING AZO DYE TECHNIQUE

1968 ◽  
Vol 36 (2) ◽  
pp. 289-297 ◽  
Author(s):  
Masando Hayashi ◽  
Tsuranobu Shirahama ◽  
Alan S. Cohen
Keyword(s):  
Electron Microscopy ◽  
Rat Liver ◽  
Dense Body ◽  
Electron Microscopic ◽  
Glucuronidase Activity ◽  
Dense Bodies ◽  
The Matrix ◽  
Matrix Control ◽  
Parenchymal Cells ◽  
Cytochemical Technique

Rat liver was fixed in formal-cacodylate-sucrose and frozen sections were incubated in a simultaneous-coupling medium containing naphthol AS-BI glucuronide as substrate and hexazonium pararosanilin as the diazo reagent. By light microscopy, the sections demonstrated ß-glucuronidase activity as red discrete granules in the pericanalicular cytoplasm and as a generalized cytoplasmic stain in the parenchymal cells. Brief treatment of sections in cold ethanol prior to incubation markedly enhanced the staining for the enzyme and made it possible to demonstrate sufficient amounts of the reaction product in sections embedded in epoxy resin following dehydration and propylene oxide treatment. Electron microscopy revealed that the reaction product was moderately electron opaque and deposited in greater amounts in the vacuolated dense bodies and occasionally in the dense bodies which did not show obvious vacuoles. In each dense body, the deposits occurred preferentially at the edge as well as in the area surrounding the vacuoles in the matrix. Control sections incubated in the presence of glucosaccharo-1:4-lactone were devoid of the reaction product. No deposits of the reaction product were found in the nucleus, mitochondria, or microbodies. The limitations of the present cytochemical technique for use in electron microscopy are briefly discussed.

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 A procedure for light and electron microscopic intracellular immunolocalization of collagen and fibronectin in rat liver.

1985 ◽  
Vol 33 (5) ◽  
pp. 407-414 ◽  
Author(s):  
B Clement ◽  
M Rissel ◽  
S Peyrol ◽  
Y Mazurier ◽  
J A Grimaud ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Type Iii Collagen ◽  
Collagen Types ◽  
Electron Microscopic ◽  
Experimental Conditions ◽  
The Matrix ◽  
Matrix Components ◽  
Indirect Immunoperoxidase

Experimental conditions have been designed that permit both extracellular and intracellular immunolocalization of various collagen types and fibronectin in rat liver. The procedure involves paraformaldehyde fixation by perfusion of the organ, use of saponin as a membrane permeabilizing agent, and visualization of the matrix components by indirect immunoperoxidase. Intracellular demonstration of collagens was particularly sensitive to the composition of the fixative and the duration of fixation. Hepatocytes contained fibronectin and types I and IV collagen, whereas fat-storing and endothelial cells evidenced type III collagen in addition. All the components were specifically located in the endoplasmic reticulum and/or the Golgi apparatus.

scholarly journals Immuno-electron-microscopic studies on the subcellular distribution of rat liver epoxide hydrolase and the effect of phenobarbitone and 2-acetamidofluorene treatment

1982 ◽  
Vol 202 (3) ◽  
pp. 677-686 ◽  
Author(s):  
F Waechter ◽  
P Bentley ◽  
M Germann ◽  
F Oesch ◽  
W Stäubli
Keyword(s):  
Electron Microscopy ◽  
Rat Liver ◽  
Subcellular Distribution ◽  
Epoxide Hydrolase ◽  
Specific Binding ◽  
Subcellular Fractions ◽  
Electron Microscopic ◽  
Immuno Electron Microscopy ◽  
Microscopic Studies ◽  
The Individual

The distribution of rat liver epoxide hydrolase in various subcellular fractions was investigated by immuno-electron-microscopy. Ferritin-linked monospecific anti-(epoxide hydrolase) immunoglobulins bound specifically to the cytoplasmic surfaces of total microsomal preparations and smooth and rough microsomal fractions as well as the nuclear envelope. Specific binding was not observed when the ferritin conjugates were incubated with peroxisomes, lysosomes and mitochondria. The average specific ferritin load of the individual subcellular fractions correlated well with the measured epoxide hydrolase activities. This correlation was observed with fractions prepared from control, phenobarbitone-treated and 2-acetamidofluorene-treated rats.

The Time Relation Between The Haemostatic Aggregation Of Platelets And Their Release Reaction In Vivo

1981 ◽  
Author(s):  
P Görög ◽  
G V R Born
Keyword(s):  
Electron Microscopy ◽  
Dense Body ◽  
Feedback Mechanism ◽  
Mesenteric Arteries ◽  
Considerable Uncertainty ◽  
Release Reaction ◽  
Dense Bodies ◽  
Aggregation Of Platelets

The release reaction of platelets has been assumed to subserve a positive feedback mechanism responsible for their aggregation in haemostasis and thrombosis. This assumption is based mainly on in vitro experiments. Considerable uncertainty remains about the contribution of the release reaction to the initiation of haemostasis in vivo. The rapidity of the process and the presence of other tissues makes it impossible to follow the reaction quantitatively in vivo by methods which permit this in vitro. We have therefore applied quantitative electron microscopy to find out how quickly the concentration of dense bodies decreases in platelets during their haemostatic aggregation. In mice, platelets were enriched in dense bodies by pretreatment with serotonin.Mesenteric arteries were incised with a sharp blade. Bleeding was stopped by a micromanipulator-operated device about 15 sec and 60 sec after the cut. The cut segments were immediately fixed in situ with glutaraldehyde and postfixed. Serial sections were made for electron microscopy. Platelets isolated from peripheral blood of the same animal were prepared similarly. Electron micrographs were projected on to a television screen and numbers of dense bodies and total platelet areas were determined by an image analysing computer. After 15 sec there were no significant differences in numbers of dense bodies in platelets from different parts of the haemostatic plugs (8.31±0.57/100 μ2 mean ± s.e.m.) and in platelets from the blood (8.93±0.38). On the other hand, after 60 sec the parts furthest from the cut contained fewer dense bodies than the nearer parts and the overall dense body number (5.86±0.05) was considerably smaller (p<0.001) than that of platelets from the blood (14.45±0.09).The results suggest that haemostatic aggregation of platelets does not initially depend on their release reaction.

scholarly journals Immunoelectron microscopic study of a new D-amino acid oxidase-immunoreactive subcompartment in rat liver peroxisomes.

1991 ◽  
Vol 39 (1) ◽  
pp. 95-102 ◽  
Author(s):  
N Usuda ◽  
S Yokota ◽  
R Ichikawa ◽  
T Hashimoto ◽  
T Nagata
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Small Area ◽  
Electron Microscopic Observation ◽  
Glycolate Oxidase ◽  
Acid Oxidase ◽  
Electron Microscopic ◽  
Amino Acid Oxidase ◽  
The Matrix ◽  
Electron Lucent

We report the presence of a new subcompartment in rat liver peroxisomal matrix in which only D-amino acid oxidase is localized and other matrix enzymes are absent. By electron microscopic observation, the rat liver peroxisome has generally been considered to consist of a single limiting membrane, an electron-dense crystalline core, and a homogeneous matrix. Immunohistochemical staining for D-amino acid oxidase by the protein A-gold technique revealed the presence of a small area in the matrix that was immunoreactive for the enzyme and was less electron-dense than the surrounding matrix. The localization of D-amino acid oxidase in this small area of the peroxisomal matrix was confirmed by immunoelectron microscopy on freeze-substituted tissues processed without chemical fixation. To analyze the characteristics of the electron-lucent area, immunoreactivity for various peroxisomal enzymes, including catalase, acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, 3-ketoacyl-CoA thiolase, L-alpha-hydroxy acid oxidase (isozyme B), and glycolate oxidase (isozyme A), was assayed. The electron-lucent area was negative for all of these. By double staining for D-amino acid oxidase and catalase, using colloidal gold particles of different sizes, these enzymes were shown to be located in separate areas in the matrix.

scholarly journals ELECTRON MICROSCOPY OF INTRANUCLEAR INCLUSIONS FOUND IN HUMAN AND RAT LIVER PARENCHYMAL CELLS

1956 ◽  
Vol 2 (4) ◽  
pp. 435-438 ◽  
Author(s):  
Ruth G. Kleinfeld ◽  
Marie H. Greider ◽  
Walter J. Frajola
Keyword(s):  
Electron Microscopy ◽  
Rat Liver ◽  
Intranuclear Inclusions ◽  
Liver Parenchymal ◽  
Parenchymal Cells

scholarly journals THE LOCALIZATION OF ACID PHOSPHATASE IN RAT LIVER CELLS AS REVEALED BY COMBINED CYTOCHEMICAL STAINING AND ELECTRON MICROSCOPY

1961 ◽  
Vol 11 (1) ◽  
pp. 47-66 ◽  
Author(s):  
S. J. Holt ◽  
R. Marian Hicks
Keyword(s):  
Electron Microscopy ◽  
Acid Phosphatase ◽  
Light Microscope ◽  
Staining Pattern ◽  
Dense Body ◽  
Butyl Methacrylate ◽  
Nuclear Staining ◽  
Heterogeneous Distribution ◽  
Dense Bodies ◽  
Before And After

Discrete localization of stain in pericanalicular granules was found in 10 µ frozen sections of formol-phosphate-sucrose-fixed liver stained by the Gomori acid phosphatase technique and examined in the light microscope. The staining patterns, before and after treatment with Triton X-100 and lecithinase, were identical with those previously reported for formol-calcium-fixed material treated in the same way, and it can be assumed that the stained granules are identical with "lysosomes." Examination in the light microscope of the staining patterns and lead penetration in fixed blocks and slices of various dimensions showed nuclear staining and other artefacts to be present, produced by the different rates of penetration of the various components of the staining medium into the tissue. A uniform pericanalicular staining pattern could be obtained, however, with slices not more than 50 µ thick, into which the staining medium could penetrate rapidly from both faces. The staining pattern produced in 50 µ slices was the same both at pH 5.0 and pH 6.2, and was not altered by subsequent embedding of the stained material in butyl methacrylate. Electron microscopy showed the fine structure of fixed 50 µ frozen slices to be well preserved, but it deteriorated badly when they were incubated in the normal Gomori medium at pH 5.0 before postfixing in osmium tetroxide. After incubation in the Gomori medium at pH 6.2, the detailed morphology was substantially maintained. In both cases lead phosphate, the reaction product, was found in the pericanalicular regions of the cell, but only in the vacuolated dense bodies and never in the microbodies. Not every vacuolated dense body contained lead, and stained and unstained bodies were sometimes seen adjacent to each other. This heterogeneous distribution of stain within a morphologically homogeneous group of particles is consistent with de Duve's suggestion (9) that there is a heterogeneous distribution of enzymes within the lysosome population. It is concluded from these investigations that the vacuolated dense bodies seen in the electron microscope are the morphological counterparts of the "lysosomes" defined biochemically by de Duve.

scholarly journals EVIDENCE FOR CHANGES IN PROTEIN POLYSACCHARIDE ASSOCIATED WITH THE ONSET OF CALCIFICATION IN CARTILAGE

1968 ◽  
Vol 39 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Victor J. Matukas ◽  
George A. Krikos
Keyword(s):  
Electron Microscopy ◽  
Osmium Tetroxide ◽  
Marked Decrease ◽  
Uranyl Acetate ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Calcified Cartilage ◽  
The Matrix ◽  
Microscopic Studies ◽  
Matrix Components

Past work has suggested that protein polysaccharide may play a role in the calcification of cartilage. Recent electron microscopic studies on noncalcified cartilage have indicated that protein polysaccharide in cartilage matrix is represented by granules associated with collagen fibers. The present work has been designed for comparison of the matrix of noncalcified cartilage to that of calcified cartilage, with particular reference to these granules. Small blocks of tibia from 16-day embryos were fixed in cacodylate-buffered glutaraldehyde and postfixed in either phosphate- or Veronal-buffered osmium tetroxide. Special care was taken to maintain the pH above 7.0 at all times. For electron microscopy the tissues were dehydrated, embedded in Epon 812, sectioned, and stained with uranyl acetate or lead citrate. A marked decrease in the size of granules in the matrix of calcified cartilage compared to noncalcified cartilage was noted. Associated with the decrease in the size of granules was a condensation of matrix components and the presence of an amorphous electron-opaque material that was not seen in noncalcified areas. These results are interpreted to represent either a drop in concentration or a change in state of protein polysaccharide with the onset of calcification in cartilage.

scholarly journals PHOSPHATASES OF EPIPHYSEAL CARTILAGE STUDIED BY ELECTRON MICROSCOPIC CYTOCHEMICAL METHODS

1971 ◽  
Vol 19 (12) ◽  
pp. 801-808 ◽  
Author(s):  
TATSUJI MATSUZAWA ◽  
H. CLARKE ANDERSON
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Plasma Membranes ◽  
Matrix Vesicles ◽  
Electron Microscopic ◽  
Previous Suggestion ◽  
Hypertrophic Zone ◽  
Dense Bodies ◽  
The Matrix ◽  
Epiphyseal Plates

The presence and distribution of alkaline phosphatase, adenosine triphosphatase (ATPase) and acid phosphatase activities in the epiphyseal plates of young mice were studied by electron microscopic cytochemical methods. Alkaline phosphatase and ATPase activities were associated with the plasma membranes of chondrocytes and with the investing membranes of matrix vesicles. These vesicles contain the earliest recognized deposits of hydroxyapatite and may promote calcification through an active process. Alkaline phosphatase and ATPase activities were greatest in the hypertrophic zone of the epiphysis, which is an area of beginning calcification. Acid phosphatase activity was demonstrable in the cytoplasm of chondrocytes in association with dense bodies which were larger than matrix vesicles and were devoid of alkaline phosphatase and ATPase activities. These cytoplasmic lysosome-like bodies were slightly more numerous in the hypertrophic zone but disappeared in the underlying zone of chondrocyte degeneration and matrix calcification. Our observations do not support a previous suggestion by others that matrix vesicles represent lysosomes. The presence of ATPase and alkaline phosphatase is compatible with an enzymatic calcium- and/or phosphate-concentrating mechanism in the matrix vesicles.

scholarly journals Isolation of a matrix that binds medial Golgi enzymes

1994 ◽  
Vol 124 (4) ◽  
pp. 405-413 ◽  
Author(s):  
P Slusarewicz ◽  
T Nilsson ◽  
N Hui ◽  
R Watson ◽  
G Warren
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Dissociation Constant ◽  
Rat Liver ◽  
Proteinase K ◽  
Apparent Dissociation Constant ◽  
Low Salt ◽  
The Matrix ◽  
Matrix Removal ◽  
Triton X

Rat liver Golgi stacks were extracted with Triton X-100 at neutral pH. After centrifugation the low speed pellet contained two medial-Golgi enzymes, N-acetylglucosaminyltransferase I and mannosidase II, but no enzymes or markers from other parts of the Golgi apparatus. Both were present in the same structures which appeared, by electron microscopy, to be small remnants of cisternal membranes. The enzymes could be removed by treatment with low salt, leaving behind a salt pellet, which we term the matrix. Removal of salt caused specific re-binding of both enzymes to the matrix, with an apparent dissociation constant of 3 nM for mannosidase II. Re-binding was abolished by pretreatment of intact Golgi stacks with proteinase K, suggesting that the matrix was present between the cisternae.

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