Demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver using a combined light- and electron-microscope procedure

1995 ◽  
Vol 27 (11) ◽  
pp. 914-922 ◽  
Author(s):  
Jiying Song ◽  
Klazina S. Bosch ◽  
Wikky Tigchelaar ◽  
Rosier J. M. Van Den Munckhof ◽  
Jacques P. M. Schellens ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Rat Liver ◽  
Nucleotidase Activity ◽  
Cryostat Sections ◽  
Unfixed Cryostat

scholarly journals Quantitative in situ analysis of xanthine oxidoreductase activity in rat liver.

1995 ◽  
Vol 43 (7) ◽  
pp. 723-726 ◽  
Author(s):  
W M Frederiks ◽  
K S Bosch ◽  
A Kooij
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Tetrazolium Salt ◽  
Xanthine Oxidoreductase ◽  
Oxidoreductase Activity ◽  
Final Reaction Product ◽  
Final Reaction ◽  
Cryostat Sections ◽  
Unfixed Cryostat

The tetrazolium salt method previously developed for the detection of xanthine oxidoreductase activity in unfixed cryostat sections has been validated for quantitative purposes. The specificity of the enzyme reaction was studied by incubating unfixed cryostat sections of rat liver in test medium containing the substrate hypoxanthine, in control medium that lacked the substrate, and in medium containing substrate and allopurinol, a specific inhibitor of xanthine oxidoreductase activity. The specific reaction rate was determined cytophotometrically by subtracting the amount of final reaction product generated in the control reaction from that formed in the test reaction. Highest specific enzyme activity in rat liver was found when the incubation medium contained 18% (w/v) polyvinyl alcohol, 100 mM phosphate buffer, pH 7.8, 0.45 mM 1-methoxyphenazine methosulfate, 5 mM tetranitro BT, and 0.5 mM hypoxanthine. Enzyme activity was present in liver parenchymal cells and in sinusoidal cells (endothelial and Kupffer cells) and was completely inhibited by allopurinol. A linear relationship was observed between the specific amount of final reaction product generated at 37 degrees C and incubation time at least up to 21 min, as well as section thickness up to 12 microns. Xanthine oxidoreductase activity, expressed as mumoles substrate converted per cm3 tissue/min, was 1.61 +/- 0.34 in pericentral areas and 1.24 +/- 0.16 in periportal areas. These values are similar to biochemical data reported in the literature. In conclusion, the tetrazolium method to detect xanthine oxidoreductase activity in unfixed cryostat sections of rat liver gives a reliable reflection of in situ activity.

scholarly journals The use of unfixed cryostat sections for electron microscopic study of D-amino acid oxidase activity in rat liver.

1992 ◽  
Vol 40 (12) ◽  
pp. 1975-1979 ◽  
Author(s):  
J P Schellens ◽  
W M Frederiks ◽  
C J Van Noorden ◽  
H Vreeling-Sindelárová ◽  
F Marx ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Oxidase Activity ◽  
Ultrastructural Level ◽  
Semipermeable Membrane ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Final Reaction Product ◽  
Cryostat Sections ◽  
Unfixed Cryostat

Unfixed cryostat sections of rat liver were incubated to demonstrate D-amino acid oxidase activity at the ultrastructural level. Incubation was performed by mounting the sections on a semipermeable membrane which was stretched over a gelled incubation medium containing D-proline as substrate and cerium ions as capture reagent for hydrogen peroxide. After an incubation period of 30 min, ultrastructural morphology was retained to such an extent that the final reaction product could be localized in peroxisomes, whereas the crystalline core remained unstained. Control incubations were performed in the absence of substrate; the lack of final reaction product in peroxisomes indicated the specificity of the reaction. We conclude that the semipermeable membrane technique opens new perspectives for localization of enzyme activities at the ultrastructural level without prior tissue fixation, thus enabling localization of the activity of soluble and/or labile enzymes.

scholarly journals Localization of uric acid oxidase activity in core and matrix of peroxisomes as detected in unfixed cryostat sections of rat liver.

1994 ◽  
Vol 42 (2) ◽  
pp. 177-183 ◽  
Author(s):  
R J Van den Munckhof ◽  
H Vreeling-Sindelárová ◽  
J P Schellens ◽  
W M Frederiks
Keyword(s):  
Uric Acid ◽  
Rat Liver ◽  
Oxidase Activity ◽  
Competitive Inhibitor ◽  
Acid Oxidase ◽  
Specific Substrate ◽  
Cytoplasmic Matrix ◽  
The Core ◽  
Cryostat Sections ◽  
Unfixed Cryostat

Because of controversial data in the literature, we studied the localization of uric acid oxidase (UAOX) activity in rat liver by light microscopy (LM) and electron microscopy (EM). UAOX is partially inactivated by aldehyde fixation and therefore we developed a technique that permits the use of unfixed cryostat sections for both LM and EM studies. Sections of rat liver were mounted on a semipermeable membrane stretched over a gelled incubation medium containing urate as specific substrate for UAOX and cerium ions to capture H2O2 produced by oxidase activity. The specificity of the reaction was checked by comparing incubations in the presence of substrate with incubations either in the absence of substrate or in the presence of substrate and 2,6,8-trichloropurine, a competitive inhibitor of UAOX. After incubation the sections were either fixed immediately for EM or visualized for LM with a second-step incubation. At the LM level, final reaction product was found in a granular form, homogeneously distributed throughout the hepatocytes. EM revealed excellent subcellular morphology and electron-dense reaction product in both the core and the matrix of peroxisomes, but not in other organelles or the cytoplasmic matrix. After incubations without substrate or with substrate and inhibitor, hardly any reaction product was found. We conclude that, because of the use of unfixed tissue, UAOX is not inactivated, which results in localization of UAOX activity not only in the core of peroxisomes but also in the peroxisomal matrix.

scholarly journals An Electron Microscope Study of Basophile Substances of Frozen-Dried Rat Liver

1958 ◽  
Vol 4 (3) ◽  
pp. 291-300 ◽  
Author(s):  
Henry Finck
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Rat Liver ◽  
Microscope Study ◽  
Electron Microscope Study ◽  
Freeze Drying ◽  
Light And Electron Microscopy ◽  
Enzyme Treatment ◽  
Dense Granules ◽  
Homogeneous Matrix

Small pieces of liver from rats subjected to different dietary regimes were fixed by freeze-drying, and postfixed by in vacuo heating and denaturation with alcohol. Specimens were digested with ribo- or deoxyribonuclease, and stained with gallocyanin-chromalum, azure II, the Feulgen procedure or alcoholic platinic tetrabromide. Some specimens were reserved as controls of the effects of enzyme treatment. Stained and unstained specimens were embedded in methacrylate and examined by light and electron microscopy. Basophilic and Feulgen-positive substances, after contact with watery reagents, were found by electron microscopy to exist as small dense granules embedded in a less dense homogeneous matrix, forming the walls of submicroscopic vacuoles. These granules were absent after digestion with nucleodepolymerases. In specimens (unstained, or stained with platinic tetrabromide) which had not passed through water, the dense (basophile) substances in nuclei and cytoplasm were found to exist, not as granules, but as ill defined submicroscopic concentrates which blended imperceptibly into the homogeneous matrix of the vacuolar walls. Objections to the use of stains for improving contrast conditions in electron microscopy of tissues are discussed, and it is concluded that the reagents do not necessarily produce the observed increases in contrast by selectively stabilizing certain structures. The concept of microsomes as pre-existing distinct morphological entities in intact (unhomogenized) cells is thought to be inconsistent with the distribution of basophile substances in frozen-dried liver.

scholarly journals Analytical study of microsomes and isolated subcellular membranes from rat liver. VI. Electron microscope examination of microsomes for cytochrome b5 by means of a ferritin-labeled antibody.

1976 ◽  
Vol 71 (2) ◽  
pp. 551-564 ◽  
Author(s):  
J Remacle ◽  
S Fowler ◽  
H Beaufay ◽  
A Amarcostesec ◽  
J Berthet
Keyword(s):  
Electron Microscope ◽  
Rat Liver ◽  
Sucrose Gradient ◽  
Analytical Study ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Microscope Examination ◽  
Group B ◽  
Isopycnic Centrifugation

The distribution of cytochrome b5 in rat liver microsomes, and in two microsomal subfractions isolated by density equilibration in a linear sucrose gradient, was studied under the electron microscope by means of a ferritin-labeled hybrid anti-cytochrome b5/anti-ferritin antibody. Results of this study show that cytochrome b5 is present in essentially all microsomal vesicles derived from endoplasmic reticulum (ER), whether rough or smooth. Thus, the dissociation of ER constituents into two groups (b and c), achieved by subfractionating microsomes by isopycnic centrifugation (Beaufay, H., A. Amar-Costesec, D. Thines-Sempoux, M. Wibo, M. Robbi, and J. Berthet. 1974. J. Cell Biol. 61:213-231), does not reflect the association of each group with distinct microsomal particles but reflects rather an enzymatic heterogeneity of the ER: the ratio of group c to group b enzymes increasing with the density and ribosome load of the particles.

scholarly journals A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method.

1975 ◽  
Vol 23 (3) ◽  
pp. 200-207 ◽  
Author(s):  
D M Boorsma ◽  
G L Kalsbeek
Keyword(s):  
Horseradish Peroxidase ◽  
Lupus Erythematosus ◽  
Cross Linking ◽  
Discoid Lupus Erythematosus ◽  
Gel Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Step Method ◽  
One Step ◽  
Cryostat Sections ◽  
Unfixed Cryostat

In this study we compared horseradish peroxidase (HRP)-labeled rabbit antihuman immunoglobulin G (IgG) conjugates, prepared by a one-step and a two-step method. Glutaraldehyde was used as a cross-linking agent. Two methods were used for removing unconjugated HRP: Sephadex G-200 gel chromatography and ammonium sulfate precipitation. The conjugates were characterized immunologically, immunochemically and enzymatically. The immunohistoenzymic properties of the conjugates were tested on unfixed cryostat sections of the skin of patients with chronic discoid lupus erythematosus. The influence of the presence of unconjugated HRP and unconjugated IgG was studied. Optimal results were obtained with conjugates prepared by a two-step method. Removing unconjugated HRPimproved the immunohistoenzymic properties of the conjugates. Conjugated and unconjugated IgG could be separated by Sephadex G-200 gel chromatography.

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