Use of epoxy resin slides for handling unfixed cryostat sections intended for histochemistry at the ultrastructural level

1983 ◽  
Vol 15 (8) ◽  
pp. 825-827 ◽  
Author(s):  
F. A. Von Bülow ◽  
P. E. Høyer
Keyword(s):  
Epoxy Resin ◽  
Ultrastructural Level ◽  
Cryostat Sections ◽  
Unfixed Cryostat

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 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.

scholarly journals Localization of GFP in Frozen Sections from Unfixed Mouse Tissues: Immobilization of a Highly Soluble Marker Protein by Formaldehyde Vapor

2003 ◽  
Vol 51 (3) ◽  
pp. 401-404 ◽  
Author(s):  
Harald Jockusch ◽  
Sylvana Voigt ◽  
Daniel Eberhard
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Striated Muscle ◽  
Host Cells ◽  
Frozen Sections ◽  
Mouse Tissues ◽  
Transplantation Experiment ◽  
Green Fluorescent ◽  
Cryostat Sections ◽  
Unfixed Cryostat

Green fluorescent protein (GFP) and its variants, such as enhanced GFP (EGFP), have been introduced into mammalian cells by transgenes, e.g., to distinguish donor from host cells after transplantation. Free GFP is extremely soluble and leaks out from liquid-covered cryostat sections so that fixation of whole organs before sectioning has been mandatory. This precludes the analysis of serial sections with respect to fixation-sensitive enzyme activities and antigens. We describe here a vapor fixation for sections from unfixed cryostat blocks of tissue that allows unrestricted enzyme and immunohistochemistry on adjacent sections, as demonstrated for cross-striated muscle and other tissues from EGFP transgenic “green mice” and for a transplantation experiment.

A Differential Stain For Neuronal Nucleoli In Unfixed Cryostat Sections

1978 ◽  
Vol 53 (4) ◽  
pp. 195-197
Author(s):  
Pierre A. Sandoz ◽  
Elisabeth Meier
Keyword(s):  
Cryostat Sections ◽  
Unfixed Cryostat

scholarly journals Radioimmunocytochemistry using a tritiated goat anti-rabbit second antibody.

1984 ◽  
Vol 32 (7) ◽  
pp. 778-782 ◽  
Author(s):  
E J Glazer ◽  
J Ramachandran ◽  
A I Basbaum
Keyword(s):  
Substance P ◽  
Epoxy Resin ◽  
Immunoglobulin G ◽  
Spinal Dorsal Horn ◽  
Ultrastructural Level ◽  
Microscopic Level ◽  
Light Microscopic Level ◽  
Spinal Dorsal ◽  
Double Label ◽  
Polyclonal Antisera

Affinity-purified goat anti-rabbit immunoglobulin G (GAR) was conjugated with (3H)-propionyl succinimidate and used to localize substance P (SP), enkephalin (ENK), and serotonin immunoreactive sites in the spinal dorsal horn and medulla of the rat and cat. Autoradiographic localization was demonstrated on paraffin, frozen, Vibratome, and 2 micron plastic sections. The latter were obtained from radiolabeled Vibratome sections that were embedded in epoxy resin. The distribution of SP, ENK, and serotonin demonstrated by radioimmunocytochemistry was comparable to that observed on semiadjacent sections using peroxidase-antiperoxidase (PAP) immunocytochemistry. The autoradiograms, however, were generated using primary antibody concentrations up to five times more dilute than concentrations used for the PAP procedure. Indirect radioimmunocytochemistry using a (3H) anti-immunoglobulin G second antibody can be used to localize a variety of monoclonal and polyclonal antisera. It is quantifiable at the light microscopic level and can be potentially used with peroxidase histochemistry to double label immunoreactive structures at the ultrastructural level.

Ultrastructural localization of carbonic anhydrase in mouse gastric parietal cells

1978 ◽  
Vol 36 (2) ◽  
pp. 532-533
Author(s):  
N. Sugai ◽  
S. Ito
Keyword(s):  
Carbonic Anhydrase ◽  
Picric Acid ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Parietal Cells ◽  
Thin Sections ◽  
Tissue Sections ◽  
Gastric Parietal Cells ◽  
Cryostat Sections ◽  
Precise Distribution

The histochemical localization of carbonic anhydrase in parietal cells has been described by a number of investigators and its presence in these cells at the ultrastructural level has been also reported. However the precise distribution of this enzyme is not clear and there are some questions regarding the validity of the histochemical reaction. In the present study, various modifications of the technique were explored and it was found that tissues fixed in buffered formaldehyde, glutaraldehyde with trinitrocresol or picric acid retained good reaction product localization of this enzyme. Cryostat sections of the fixed tissues were treated with solutions recommended by Hannson. Incubation times that were most favorable 5 to 10 min for light microscopy and 8 to 15 min for electron microscopy. For ultrastructural observations of thin sections, it was found to be important that the reacted tissue sections were post osmicated with 1% osmium tetroxide in 1. 5% potassium ferrocyanide or with aqueous 1% 0s04 for only 3 to 5 min.

scholarly journals A histochemical procedure for light microscopic demonstration of xanthine oxidase activity in unfixed cryostat sections using cerium ions and a semipermeable membrane technique.

1993 ◽  
Vol 41 (5) ◽  
pp. 667-670 ◽  
Author(s):  
W M Frederiks ◽  
F Marx
Keyword(s):  
Xanthine Oxidase ◽  
Oxidase Activity ◽  
Semipermeable Membrane ◽  
Myeloperoxidase Activity ◽  
Final Reaction Product ◽  
Final Reaction ◽  
Xanthine Oxidase Activity ◽  
Membrane Technique ◽  
Cryostat Sections ◽  
Unfixed Cryostat

Xanthine oxidoreductase exists in two functionally distinct forms. Under normal conditions, the larger part of the enzyme occurs as an NAD(+)-dependent dehydrogenase form which produces NADH and urate. The dehydrogenase can be transformed under various (patho)physiological conditions to an oxygen-dependent oxidase form which produces oxygen radicals and/or hydrogen peroxide and urate. Tetrazolium salts are used to demonstrate the total activity of both the dehydrogenase and the oxidase form of the enzyme. We have developed a procedure to detect the oxidase form only in unfixed cryostat sections with the use of cerium on the basis of the semipermeable membrane technique. The incubation medium contained hypoxanthine as substrate, cerium ions, and sodium azide to inhibit catalase and peroxidase activity. In a second-step reaction, diaminobenzidine was polymerized in the presence of cobalt ions by decomposition of cerium perhydroxide. Large amounts of final reaction product were found in milk droplets in the acini of lactating bovine mammary gland, whereas milk-secreting epithelial cells contained hardly any final reaction product. In rat duodenum, enzyme activity was found in the cytoplasm of enterocytes and goblet cells but not in the mucus. Control reactions performed in the absence of substrate or in the presence of substrate and allopurinol, a specific inhibitor of xanthine oxidase, were completely negative in both tissues, with the exception of polymorphonuclear leukocytes in the lamina propria of duodenum. The positive nonspecific reaction in these cells was caused by myeloperoxidase activity. We conclude that the present method is specific for the detection of xanthine oxidase activity. Moreover, conversion of the dehydrogenase form into the oxidase form can be prevented by omission of chemical fixation of the tissue in the present procedure.

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.

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