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 Relationship between peroxisomes and endoplasmic reticulum investigated by combined catalase and glucose-6-phosphatase cytochemistry.

1981 ◽  
Vol 29 (11) ◽  
pp. 1263-1272 ◽  
Author(s):  
H Shio ◽  
P B Lazarow
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Rat Liver ◽  
Reaction Products ◽  
Lead Phosphate ◽  
Electron Microscopic ◽  
Phosphatase Cytochemistry ◽  
Electron Microscopic Cytochemistry ◽  
Cellular Compartments

The theoretical advantages of electron microscopic cytochemistry were utilized to look for evidence of possible connections between peroxisomes and the endoplasmic reticulum in rat liver. Established cytochemical procedures for catalase (peroxisomes) and glucose-6-phosphatase (endoplasmic reticulum) were carried out, and evidence was sought of diffusion of reaction products between the organelles. No such diffusion was observed: lead phosphate was found in the endoplasmic reticulum and in the nuclear envelope but not in peroxisomes; oxidized diaminobenzidine (DAB) was seen only in peroxisomes. In addition, both types of cytochemistry were carried out on the same tissue. The two kinds of reaction product could be distinguished by virtue of their different electron opacities. No mixing of the two reaction products was observed. These results do not support the hypothesis that peroxisomes and endoplasmic reticulum may be connected; rather, they support the idea that the two organelles exist as separate cellular compartments.

Electron microscopic tomography of rat-liver mitochondria and their interactions with the endoplasmic reticulum

BioFactors ◽  
1998 ◽  
Vol 8 (3-4) ◽  
pp. 225-228 ◽  
Author(s):  
Carmen A. Mannella ◽  
Karolyn Buttle ◽  
Bimal K. Rath ◽  
M. Marko
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Electron Microscopic

scholarly journals 3,4,3,4'-Tetrachlorobiphenyl-Induced Effects in the Rat Liver. II. Electron Microscopic Autoradiographic Localization of H-TCB

1989 ◽  
Vol 17 (4_part_2) ◽  
pp. 782-788 ◽  
Author(s):  
Stephen K. Durham ◽  
Abraham Brouwer
Keyword(s):  
Endoplasmic Reticulum ◽  
Endothelial Cell ◽  
Rat Liver ◽  
Kupffer Cell ◽  
Liver Cell ◽  
Morphologic Change ◽  
Cell Type ◽  
Sequential Order ◽  
Electron Microscopic ◽  
Morphological Alterations

Recent results (3) indicate that 200 mg 3,4,3′,4′-tetrachlorobiphenyl induces hepatomegaly accompanied by significant decreases in serum and hepatic retinoid content and hepatocyte morphologic alterations of proliferated and vesiculated endoplasmic reticulum and megamitochondria with paracrystalline inclusions. There was also an associated change in the number, size, and distribution of lipid droplets in hepatocytes and fat-storing cells. Electron microscopic autoradiographic techniques were utilized to determine the cellular and subcellular distribution of 3H-3,4,3′,4′-tetrachlorobiphenyl (3H-TCB) in the adult rat liver and determine if there is any relationship between subcellular morphologic change and radiolabel localization. Adult female WAG/Rij rats received a single intraperitoneal injection of 200 mg TCB/kg containing 1.85 mCi of 3H-TCB and were sacrificed at 1, 3, 7, and 14 days following exposure. The vast majority of 3H-TCB-derived radioactivity was located in the hepatocyte at all time points examined, ranging from 79–86% of the total number of autoradiographic grains counted over the liver cells. Sequential order of radiolabel localization per liver cell type at 1, 3, and 7 days was hepatocyte > > > Kupffer cell > fat-storing cell > endothelial cell. At day 14, the sequential order of radiolabel localization per liver cell type was hepatocyte > > > fat-storing cell > Kupffer cell > endothelial cell, which indicates that there was some shift movement of label over time. The lipid droplet, mitochondria, and endoplasmic reticulum were the subcellular structures or organelles of hepatocytes having the highest number of 3H-TCB-derived grains at all time periods examined. The predominant morphological alterations induced following TCB intoxication were observed in these organelles. The results of this study suggests that there is an association between TCB localization and morphologic change induced in mitochondria and endoplasmic reticulum of hepatocytes following TCB exposure.

scholarly journals Fluorophosphate-sensitive esterases in mammalian liver: the radioautographic localization and measurement of fluorophosphate-reactive sites in adult rat liver.

1980 ◽  
Vol 28 (6) ◽  
pp. 533-542 ◽  
Author(s):  
G C Budd ◽  
E A Barnard ◽  
C Porter ◽  
S A Mattimoe
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Rat Liver ◽  
Active Sites ◽  
Smooth Endoplasmic Reticulum ◽  
Liver Volume ◽  
Male Rat ◽  
Background Concentration ◽  
Experimental Conditions ◽  
Total Liver

Fluorophosphate-reactive (FPR) sites in the adult male rat liver, tentatively identified as esterase active centers, were localized and measured using the combined techniques of quantitative electron microscope radioautography and morphometric analysis with the light and electron microscope. The FPR sites were measured in liver which had been prefixed by perfusion with 1.5% glutaraldehyde and reacted with 10(-4) M tritium-labeled diisopropyl fluorophosphate (3H-DFP). Under the experimental conditions 64-67% of the esterase activity in fresh liver was retained for reaction with the 3H-DFP, which is known to bind irreversibly to the active sites of certain esterases. In light and electron microscope radioautographs the developed silver grains were concentrated over the cytoplasm of hepatocytes. A low concentration occurred over erythrocytes. All other areas in the liver had a concentration of grains resembling the background concentration. Quantitative measurements of grain density in the electron microscope radioautographs revealed the highest density, after correcting for radiation spread, in cytoplasmic granules (mainly cytolysomes). The grain densities over the rough and smooth endoplasmic reticulum and associated structures were also equal to or above the average hepatocyte grain density. Due to the large fractional volume of endoplasmic reticulum per hepatocyte (58% of cell volume) and the fraction of the liver occupied by hepatocytes (79% of liver volume) the majority of FPR sites in the liver occurred in the rough and smooth endoplasmic reticulum and associated structures. The average numbers of FPR sites were calculated per micrometer3 of hepatocyte (5.0 x 10(5) sites/micrometer3) and per unit volume of each significantly labeled organelle. In addition, the number of FPR sites per hepatocyte (2.5 X 10(9) sites/cell), per cm3 liver (4.1 X 10(17) sites/cm3) and in the total liver of an average 100 g male rate (2.2 X 10(18) sites/total liver) were also calculated.

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.

A Rapidly Sedimenting Fraction of Rat Liver Endoplasmic Reticulum

1973 ◽  
Vol 13 (2) ◽  
pp. 447-459 ◽  
Author(s):  
J. A. LEWIS ◽  
J. R. TATA
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Close Association ◽  
Balance Sheet ◽  
Subcellular Fractionation ◽  
Electron Microscopic ◽  
Microsomal Preparation ◽  
Low Speed ◽  
Microscopic Studies

Balance-sheet experiments carried out to account for the distribution of endoplasmic reticulum fragments during subcellular fractionation of rat liver showed that a large proportion of these fragments are present in the pellets of low-speed centrifugation. Using glucose-6-phosphatase and RNA as markers we found that approximately 50% of the fragments of endoplasmic reticulum sedimented in the pellet of a 640-g centrifugation, 10% in that of a 6000-g centrifugation and 35% in the pellet of a 105000-g centrifugation. Starvation of the animals before use did not alter this distribution, nor did the use of more vigorous homogenization conditions. We have developed a procedure for removing nuclei and erythrocytes from the material sedimenting at 640g to give a fraction (rapidly sedimenting ER fraction or RS-ER) similar to the standard microsomal preparation. Centrifugation of this RS-ER fraction over 1.3 M sucrose yields subfractions of high and low RNA content analogous to the rough and smooth microsomal fractions. Electron-microscopic studies showed that, whereas the rough microsomal fraction consisted of ribosome-studded vesicles of varying size and content density, the rough RS-ER fraction contained a mixture of mitochondria and double lamellar membranes with ribosomes attached. These double lamellar membranes closely resemble the endoplasmic reticulum of intact rat liver. The double lamellar membranes are frequently observed grouped in stacks and in close association with the mitochondria. The significance of the association between endoplasmic reticulum and mitochondria of the RS-ER fraction and the relation between it and the standard microsomal preparation are discussed.

Secretion of collagen types I and II by epithelial and endothelial cells in the developing chick cornea demonstrated by in situ hybridization and immunohistochemistry

Development ◽  
1988 ◽  
Vol 103 (1) ◽  
pp. 27-36 ◽  
Author(s):  
M. Hayashi ◽  
Y. Ninomiya ◽  
K. Hayashi ◽  
T.F. Linsenmayer ◽  
B.R. Olsen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Epithelial Cells ◽  
Type I Collagen ◽  
Type I ◽  
Basal Cells ◽  
Collagen Types ◽  
Electron Microscopic ◽  
Specific Mrnas

Cells involved in the synthesis of collagen types I and II in the cornea of developing chick embryos have been studied by using in situ hybridization and immunohistochemistry. Corneas processed for in situ hybridization with the type I and II collagen probes demonstrated specific mRNAs in the epithelium of embryos at stage 18 with an increase at stages between 26 and 31, and then gradual decrease to the background level in the next several days. In the endothelium, a small amount of specific mRNA was recognized through these stages. In the stroma, only sections hybridized with the type I probe demonstrated mRNA in fibroblasts. Immunostaining demonstrated specific collagen types in the stroma at sites which were closely associated with cells containing specific mRNAs. Both collagens type I and II were present beneath the epithelium as narrow bands at stage 18; as the thicker primary stroma at stages 20 and 26; and as subepithelial, subendothelial and stromal staining at stage 31. Thereafter, type I collagen was increased in the stroma but it was also noted in the subepithelial and, to a lesser degree, subendothelial regions, whereas type II collagen was gradually confined to the subendothelial matrix. Electron microscopic examination of sections from 5-day-old (stage-27) embryo corneas using antibodies against the carboxyl propeptides of type I and II procollagens revealed the presence of these procollagens within the cisternae of the endoplasmic reticulum and Golgi vesicles in both epithelial and endothelial cells. In the epithelial cells both the periderm and basal cells contained these procollagens within the cytoplasmic organelles. These results indicate that not only the epithelial cells, but also the endothelial cells secrete collagen types I and II during the formation of the primary corneal stroma and for several days after invasion of fibroblasts.

scholarly journals Glucagon stimulation of ruthenium red-insensitive calcium ion transport in developing rat liver

1981 ◽  
Vol 194 (2) ◽  
pp. 541-549 ◽  
Author(s):  
P H Reinhart ◽  
F L Bygrave
Keyword(s):  
Endoplasmic Reticulum ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Adult Life ◽  
Ruthenium Red ◽  
Enzyme Analysis ◽  
Marker Enzyme ◽  
Transport Activity ◽  
Electron Microscopic ◽  
Metabolic Role

The maturation of glucagon-stimulated Ruthenium Red-insensitive Ca2+-transport activity was determined in livers of rats ranging in age from 5 days preterm to 10 weeks of adult life. Previous indications are that this activity is confined to vesicles derived mainly from the endoplasmic reticulum. Perinatal-rat liver contains near-adult values of Ruthenium Red-insensitive Ca2+-transport activity, and exhibits large transient increases in the rate of this activity at two stages of development, immediately after birth, and at 2-5 days after birth. The administration of glucagon to foetal rats, at developmental stages after 19.5 days of gestation (2.5 days before birth), results in a large stable increase (greater than 100%) of Ca2+-transport activity in a subsequently isolated ‘heavy’ microsomal fraction. That this fraction was enriched in vesicles derived from the rough endoplasmic reticulum was indicated by both an electron-microscopic examination and a marker-enzyme analysis of the subcellular fractions. The administration of glucagon into newborn animals only hours old does not enhance further the initial rate of Ca2+-transport activity, and from day 1 to 10 weeks after birth the administration of the hormone results in the moderate enhancement of Ca2+ transport. Experiments with cyclic AMP and inhibitors of phosphodiesterase activity suggest that cyclic AMP plays a key role in the enhancement by glucagon of Ruthenium Red-insensitive Ca2+ transport, and arguments are presented that this transport system has an important metabolic role in the redistribution of intracellular Ca2+ in liver tissue.

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