scholarly journals Cytochemical localization of ornithine decarboxylase with rhodamine or biotin-labeled alpha-difluoromethylornithine. An example for the use of labeled irreversible enzyme inhibitors as cytochemical markers.

1981 ◽  
Vol 29 (6) ◽  
pp. 687-692 ◽  
Author(s):  
G M Gilad ◽  
V H Gilad
Keyword(s):  
Ornithine Decarboxylase ◽  
Enzyme Inhibitors ◽  
Ultrastructural Localization ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
Tissue Sections ◽  
Microscopic Studies ◽  
Fluorescence Cytochemistry ◽  
Rhodamine B Isothiocyanate ◽  
Developing Rat

The present work describes a new method for cytochemical localization of enzymes using ornithine decarboxylase (ODC) as an example. The method is based on the preservation of the characteristic-specific and irreversible binding of the inhibitor alpha-difluoromethylornithine (alpha-DFMO) following its conjugation to "label" molecules. The inhibitor has been conjugated to the fluorescent molecule rhodamine-B-isothiocyanate, and its localization in tissue sections was detected directly by fluorescence cytochemistry. Alternatively, alpha-DFMO has been conjugated to biotin and its cytochemical localization determined indirectly following its binding with avidin conjugated to horseradish peroxidase (HRP) and visualization of the HRP reaction product. Both labeled inhibitor molecules were successfully localized cytochemically within specific cells of the developing rat cerebellum and rat liver following thioacetamide injection where ODC activity is greatly enhanced. This novel technique should be of general application 1) in other tissues, 2) for other enzymes, and 3) in electron microscopic studies for ultrastructural localization of the enzyme.

scholarly journals Ultrastructural cytochemical localization of uricase in peroxisomes of rat liver.

1986 ◽  
Vol 34 (2) ◽  
pp. 159-165 ◽  
Author(s):  
S Angermüller ◽  
H D Fahimi
Keyword(s):  
Rat Liver ◽  
Specific Inhibitor ◽  
Ultrastructural Localization ◽  
Cerium Chloride ◽  
Light Microscopic Level ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
Microscopic Studies ◽  
Parenchymal Cells ◽  
Sodium Urate

Ultrastructural localization of uricase (urate: oxygen oxidoreductase, E.C.1.7.3.3.) in rat liver parenchymal cells has been studied with the cerium technique. The cerous ions react with H2O2 generated by the activity of the enzyme in the presence of urate, forming the electron-dense reaction product of cerous perhydroxide. Tissue fixation is carried out by perfusion for 5 min with a low concentration (0.25%) of glutaraldehyde. Since in a biochemical assay it was found that the activity of uricase determined in Trismaleate buffer is substantially weaker than in the Pipes buffer, the classical medium of Briggs et al. (6) was modified, and the latter buffer was substituted for the Trismaleate. Vibratome sectons are incubated at 37 degrees C for 60 min in 0.1 M Pipes buffer, pH 7.8, containing 3 mM cerium chloride and 0.1 mM sodium urate. Under these conditions, the reaction product is localized in the crystalline cores of hepatic peroxisomes. The intensity of the staining is dependent on the concentration of the substrate and the incubation time. In control preparations incubated without urate or with 2,6,8-trichloropurine, a specific inhibitor of uricase, staining is almost completely abolished. In sections incubated with 5 mM cerium and 0.1 mM sodium urate, fine granules with a distribution corresponding to peroxisomes are also visible at the light microscopic level. This latter observation is invaluable for correlative light and electron microscopic studies.

Peroxidase Localization in Hartmanella Trophozoites

1971 ◽  
Vol 29 ◽  
pp. 346-347 ◽  
Author(s):  
George E. Childs ◽  
Joseph H. Miller
Keyword(s):  
Hydrogen Peroxide ◽  
Ultrastructural Localization ◽  
Pathogenic Strain ◽  
Oxidative Enzymes ◽  
Differential Centrifugation ◽  
Electron Microscopic ◽  
Microscopic Studies ◽  
The Subject ◽  
Axenic Cultures

Biochemical and differential centrifugation studies have demonstrated that the oxidative enzymes of Acanthamoeba sp. are localized in mitochondria and peroxisomes (microbodies). Although hartmanellid amoebae have been the subject of several electron microscopic studies, peroxisomes have not been described from these organisms or other protozoa. Cytochemical tests employing diaminobenzidine-tetra HCl (DAB) and hydrogen peroxide were used for the ultrastructural localization of peroxidases of trophozoites of Hartmanella sp. (A-l, Culbertson), a pathogenic strain grown in axenic cultures of trypticase soy broth.

The effects of an antiserum to the contractile proteins of the heart on the developing rat embryo

Development ◽  
1971 ◽  
Vol 25 (2) ◽  
pp. 203-212
Author(s):  
Colin L. Berry
Keyword(s):  
Rat Heart ◽  
Contractile Proteins ◽  
Degenerative Changes ◽  
Foetal Death ◽  
Rat Embryo ◽  
Electron Microscopic ◽  
Microscopic Studies ◽  
Specific Reactivity ◽  
Developing Rat ◽  
Isolated Rat

An antiserum with specific reactivity against the contractile proteins of the rat heart has been raised in the rabbit. Fractions of the serum have been shown to enter the isolated rat embryo by electron-microscopic studies with ferritin, where they bind to the myocardium, producing degenerative changes. Both IgG and IgM fractions are toxic, producing foetal death in a large proportion of explanted embryos.

scholarly journals Immunoelectron microscopic localization of estrogen receptor with monoclonal estrophilin antibodies.

1985 ◽  
Vol 33 (9) ◽  
pp. 915-924 ◽  
Author(s):  
M F Press ◽  
N A Nousek-Goebl ◽  
G L Greene
Keyword(s):  
Estrogen Receptor ◽  
Ultrastructural Localization ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Nuclear Staining ◽  
Cytoplasmic Localization ◽  
Electron Microscopic ◽  
Tissue Sections ◽  
Receptor Antibodies ◽  
Microscopic Techniques

The recent production of a series of monoclonal estrophilin (estrogen receptor) antibodies recognizing estrogen receptor derived from a wide variety of animals and target tissues permits the development of immunoelectron microscopic techniques for identifying estrogen receptor. We have determined suitable conditions for the ultrastructural localization of estrogen receptor in tissue sections. Localization of receptor was observed in the euchromatin, but not in the marginated heterochromatin or nucleoli of epithelial and stromal nuclei of human endometrium. Competition studies indicate that only estrogen receptor specifically inhibits nuclear staining. The absence of any specific cytoplasmic localization at the electron-microscopic level is consistent with earlier light-microscopic observations and suggests that the majority of the cellular pool of estrophilin exists in the nucleus of hormone-responsive cells.

Sponge secondary metabolites: biochemical and ultrastructural localization of the antimitotic agent avarol in Dysidea avara.

1986 ◽  
Vol 34 (12) ◽  
pp. 1687-1690 ◽  
Author(s):  
W E Müller ◽  
B Diehl-Seifert ◽  
C Sobel ◽  
A Bechtold ◽  
Z Kljajić ◽  
...  
Keyword(s):  
Ultrastructural Localization ◽  
Inhibitory Effect ◽  
Storage Site ◽  
Electron Microscopic ◽  
Antimitotic Agent ◽  
Transmission Electron ◽  
Cytoplasmic Vesicles ◽  
Transmission Electron Microscopic ◽  
Microscopic Studies ◽  
Dysidea Avara

The secondary metabolite avarol, a potent cytostatic and antibacterial sesquiterpenoid hydroquinone, is present in large amounts only in the sponge Dysidea avara (2.7 g avarol/1 kg of fresh material). The present study was designed to determine the storage site of this compound within the organism. Light and transmission electron microscopic studies revealed that avarol is probably stored only in spherular cells. The compound is compartmented in intracellular cytoplasmic vesicles in a paracrystalline form, and therefore can have no inhibitory effect on the sponge cells. Quantitative analysis utilizing high-pressure liquid chromatography revealed that avarol is present at a concentration of 3.2 micrograms/10(6) spherular cells. It appears that avarol is released from the cells into the extracellular space in a merocrine manner. We suggest that it is involved in regulating the bacteria with which the sponge is symbiotically associated.

Ultrastructure of Oviduct Epithelium of the Porcine in Estrus

1971 ◽  
Vol 29 ◽  
pp. 512-513
Author(s):  
R. K. Nayak ◽  
D. R. Zimmerman
Keyword(s):  
Intercellular Junctions ◽  
Uranyl Acetate ◽  
Secretory Process ◽  
Osmic Acid ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Tissue Sections ◽  
Oviduct Epithelium ◽  
Microscopic Studies ◽  
Cyclic Changes

Cyclic changes of porcine oviduct epithelium studied by light microscopy were first reported by Snyder in 1923. UltrastructuraI features of the porcine oviduct epithelium have not yet been described. Electron microscopic studies of oviduct epithelium have been reported for only a few species. These reports have been recently reviewed by Nilsson and Relnius. This report describes the fine structure of, the oviduct epithelium and attempts to elucidate the mechanism of ciliogenesis and the secretory process in the porcine during estrus.Tissue sections from the fimbria and ampulla were fixed in cold 3% cacodylate buffered glutaraldehyde (pH 7.4), post-fixed in 1% osmic acid and embedded in Epon. Ultra-thin sections were stained with uranyl acetate and lead citrate and examined in a RCA 3-G electron microscope operated at 100 kv.The epithelium of the tubal mucosa consists of secretory and ciliated cells. The cells are columnar and rest on a common basement membrane, which is about 50 mμ thick. The distal or free borders of the surface epithelial cells possess few irregular microvilli. The membranes of adjacent cells show tight intercellular junctions and macula adhaerentes (Figs. 1, 2).

scholarly journals Concurrent cytochemical localization of adenylate cyclase and peroxidase in the developing rat submandibular gland.

1977 ◽  
Vol 25 (11) ◽  
pp. 1207-1212 ◽  
Author(s):  
L S Cutler ◽  
A Mooradian ◽  
C Christian
Keyword(s):  
Adenylate Cyclase ◽  
Submandibular Gland ◽  
Secretory Granules ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
Fixed Tissue ◽  
Fetal Rat ◽  
Endogenous Peroxidase ◽  
Rat Submandibular Gland ◽  
Developing Rat

An electron microscopic histochemical technique for the concurrent localization of adenylate cyclase and endogenous peroxidase is described. The procedure involves incubation of glutaraldehyde fixed tissue in adenylate cyclase medium followed by washing and incubation in 3,3'-diaminobenzidine tetrahydrochloride medium to demonstrate peroxidase activity. Adenylate cyclase was localized at the cell surface of the tissue investigated (20 day fetal rat submandibular gland) while peroxidase was localized in the rough endoplasmic reticulum and secretory granules of some cells. Biochemical and histochemical controls indicate that the procedure is valid. The potential use of this procedure and variations of the procedure are discussed.

scholarly journals Application of the FITC-anti-FITC-gold system to ultrastructural localization of antigens.

1991 ◽  
Vol 39 (12) ◽  
pp. 1725-1728 ◽  
Author(s):  
G J van Dam ◽  
B J Bogitsh ◽  
J A Fransen ◽  
D Kornelis ◽  
R J van Zeyl ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Fluorescein Isothiocyanate ◽  
High Specificity ◽  
Ultrastructural Localization ◽  
Good Alternative ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Protein Conjugates ◽  
Specificity And Sensitivity ◽  
Microscopic Studies

We report the application of a fluorescein isothiocyanate (FITC)-anti-FITC method to localize antigens at the ultrastructural level. In the systems studied, the anti-FITC-based detection method displays high specificity and sensitivity. These observations, combined with ease of production and with availability of FITC-protein conjugates, suggest that the FITC-anti-FITC method is a good alternative to presently used methods and is widely applicable to immunochemical and immunocytochemical procedures. The same preparation and protocol can be used for light and electron microscopic studies, thereby reducing possible artifacts introduced if different procedures are used. In the present study, two systems were used to test the method. One system used an FITC-labeled monoclonal antibody (MAb) to schistosome circulating cathodic antigen. In this system, the label was detected in the gut of adult Schistosoma mansoni by an anti-FITC MAb conjugated to 10-nm gold particles. The second system used human IgM antibodies pooled from patients infected with Schistosoma mansoni. In this system detection was accomplished using an anti-human IgM-FITC conjugate followed by the anti-FITC-Au antibody conjugate.

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