Cytochemical localization of glutaraldehyde-resistant NAD(P)H-oxidase in rat hepatocytes

The intracellular localization of beta-NADPase in rat hepatocytes and Kupffer cells has been studied and compared with the pattern of TPPase in these cells. The reaction product for beta-NADPase is present in some but not all hepatocytes in two cisternae on the trans aspect of the Golgi apparatus. It is absent from the trans-most lamella and the GERL of hepatocytes. TPPase, on the other hand, is limited to the first Golgi cisterna on the trans aspect with sprinkles of reaction product in the second lamella. Considering that TPPase is a marker of the trans Golgi lamella and hepatocyte Golgi stacks contain usually 2-4 lamellae, our observations suggest that beta-NADPase is localized in the trans as well as in the intermediate Golgi lamellae of liver parenchymal cells. In Kupffer cells, the reaction product for both beta-NADPase and TPPase was found in some but not in all cells. The enzyme beta-NADPase was localized in the rigid lamella and the tubulovacuolar system of GERL. This pattern differed significantly from that for TPPase, which was found in 2-3 cisternae at the trans aspect of the Golgi complex in Kupffer cells. These observations demonstrate the difference in the localization of beta-NADPase in hepatocytes and Kupffer cells. Such differences should be taken into consideration in studies of Golgi fractions, when phosphatase reactions are used as specific markers of Golgi components.

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Cytochemical localization of Na+,K+-ATPase in rat hepatocytes

Gastroenterology ◽

10.1016/0016-5085(78)93665-x ◽

1978 ◽

Vol 74 (5) ◽

pp. 1169 ◽

Cited By ~ 1

Author(s):

B.L. Blitzer ◽

J.L Boyer

Keyword(s):

Rat Hepatocytes ◽

Cytochemical Localization

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Cytochemical localization of peroxisomes in rat hepatocytes at early stages of fetal development

Ultramicroscopy ◽

10.1016/0304-3991(83)90376-5 ◽

1983 ◽

Vol 12 (1-2) ◽

pp. 123-124

Author(s):

S. Stefanini ◽

M.P. CerùArgento

Keyword(s):

Fetal Development ◽

Rat Hepatocytes ◽

Cytochemical Localization ◽

Early Stages

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A cytochemical stain for glutathione in rat hepatocytes cultured on plastic.

Journal of Histochemistry & Cytochemistry ◽

10.1177/35.2.2432118 ◽

1987 ◽

Vol 35 (2) ◽

pp. 271-274 ◽

Cited By ~ 15

Author(s):

A Larrauri ◽

P López ◽

M J Gómez-Lechón ◽

J V Castell

Keyword(s):

Azo Dye ◽

Room Temperature ◽

Cultured Cells ◽

Rat Hepatocytes ◽

Thiol Groups ◽

Cytochemical Localization ◽

Sh Groups ◽

Acetone Water ◽

Mercury Orange ◽

Orange Fluorescence

Thiol groups of glutathione react with the organomercurial azo dye mercury orange at a faster rate than with -SH groups of proteins. This property makes possible visualization of glutathione in cells without appreciable interference from other -SH groups. To render this method useful for cytochemical localization of glutathione in plastic cultured cells, it was necessary to adapt this reaction to the specific characteristics of the biological samples to be assayed. First, the choice of a solvent that would allow a convenient solubility of the dye and at the same time be compatible with the plastic culture plate was crucial. Second, to avoid diffusion of glutathione out of the cell the procedure for staining cells was also important. Satisfactory results were obtained after 30-40 sec reaction with 50 microM mercury orange in acetone/water 9:1, v/v, at room temperature. Glutathione-mercury orange complexes exhibited orange fluorescence on excitation with blue light. No diffusion of glutathione out of the cells was observed, and the hepatocytes stained with the dye showed orange fluorescence which paralleled their glutathione content.

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Immunoenzymatic demonstration of the simultaneous presence of transferrin, hemopexin and albumin in a same hepatocyte and their ultrastructural localization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081309 ◽

1978 ◽

Vol 36 (2) ◽

pp. 88-89

Author(s):

M. Kraemer ◽

J. Foucrier ◽

J. Vassy ◽

M.T. Chalumeau

Keyword(s):

Plasma Proteins ◽

Rat Hepatocytes ◽

Ultrastructural Localization ◽

Carrier Proteins ◽

Normal Cells ◽

Adult Rat ◽

Adult Rat Hepatocytes ◽

Monospecific Antisera ◽

Different Levels

Some authors using immunofluorescent techniques had already suggested that some hepatocytes are able to synthetize several plasma proteins. In vitro studies on normal cells or on cells issued of murine hepatomas raise the same conclusion. These works could be indications of an hepatocyte functionnal non-specialization, meanwhile the authors never give direct topographic proofs suitable with this hypothesis.The use of immunoenzymatic techniques after obtention of monospecific antisera had seemed to us useful to bring forward a better knowledge of this problem. We have studied three carrier proteins (transferrin = Tf, hemopexin = Hx, albumin = Alb) operating at different levels in iron metabolism by demonstrating and localizing the adult rat hepatocytes involved in their synthesis.Immunological, histological and ultrastructural methods have been described in a previous work.

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Initial Polymerization Sites Indicated During Mitochondrial Oxidation of Diaminobenzidine after Toluene Treatment

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079498 ◽

1977 ◽

Vol 35 ◽

pp. 408-409

Author(s):

W. A. Shannon ◽

M. A. Matlib

Keyword(s):

Membrane Permeability ◽

Cytochrome Oxidase ◽

Rat Liver ◽

Precise Definition ◽

Cytochemical Localization ◽

Oxidative Reaction ◽

Mitochondrial Oxidation ◽

Definition Of ◽

Exogenous Substrates

Numerous studies have dealt with the cytochemical localization of cytochrome oxidase via cytochrome c. More recent studies have dealt with indicating initial foci of this reaction by altering incubation pH (1) or postosmication procedure (2,3). The following study is an attempt to locate such foci by altering membrane permeability. It is thought that such alterations within the limits of maintaining morphological integrity of the membranes will ease the entry of exogenous substrates resulting in a much quicker oxidation and subsequently a more precise definition of the oxidative reaction.The diaminobenzidine (DAB) method of Seligman et al. (4) was used. Minced pieces of rat liver were incubated for 1 hr following toluene treatment (5,6). Experimental variations consisted of incubating fixed or unfixed tissues treated with toluene and unfixed tissues treated with toluene and subsequently fixed.

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High-resolution scanning electron microscopy (HRSEM) of mitochondria from various rat tissues

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102158 ◽

1988 ◽

Vol 46 ◽

pp. 14-15

Author(s):

P.J. Lea ◽

M.J. Hollenberg

Keyword(s):

Electron Microscopy ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Rat Hepatocytes ◽

Three Dimensions ◽

Objective Lens ◽

Rat Tissues ◽

Thin Sections ◽

Reconstruction Methods ◽

Scanning Electron

Our current understanding of mitochondrial ultrastructure has been derived primarily from thin sections using transmission electron microscopy (TEM). This information has been extrapolated into three dimensions by artist's impressions (1) or serial sectioning techniques in combination with computer processing (2). The resolution of serial reconstruction methods is limited by section thickness whereas artist's impressions have obvious disadvantages.In contrast, the new techniques of HRSEM used in this study (3) offer the opportunity to view simultaneously both the internal and external structure of mitochondria directly in three dimensions and in detail.The tridimensional ultrastructure of mitochondria from rat hepatocytes, retinal (retinal pigment epithelium), renal (proximal convoluted tubule) and adrenal cortex cells were studied by HRSEM. The specimens were prepared by aldehyde-osmium fixation in combination with freeze cleavage followed by partial extraction of cytosol with a weak solution of osmium tetroxide (4). The specimens were examined with a Hitachi S-570 scanning electron microscope, resolution better than 30 nm, where the secondary electron detector is located in the column directly above the specimen inserted within the objective lens.

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Cytochemical Localization of Polysaccharides in Diplodia Maydis With Thiosemicarbazide : Evaluation of Three Fixatives For Pa:Tsc:Os Reaction

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100090221 ◽

1976 ◽

Vol 34 ◽

pp. 48-49 ◽

Cited By ~ 1

Author(s):

Judith A. Murphy ◽

Mary R. Thompson ◽

A.J. Pappelis

Keyword(s):

Reaction Center ◽

Osmium Tetroxide ◽

Periodic Acid ◽

Cytochemical Localization ◽

Thin Sections ◽

Selective Reaction ◽

Amorphous Character

In an attempt to identify polysaccharide components in thin sections of D. maydis, procedures were employed such that a PAS localization could be carried out. Three different fixatives were evaluated ie. glutaraldehyde, formaldehyde and paraformaldehyde. These were used in conjunction with periodic acid (PA), thiosemicarbazide(TSC), and osmium tetroxide(Os) to localize polysaccharides in V. maydis using a pre-embedded reaction procedure. Polysaccharide localization is based on the oxidation of vic-glycol groups by PA, and the binding of TSC as a selective reaction center for the formation of osmium black. The reaction product is sufficiently electron opaque, insoluble in lipids, not altered when tissue is embedded, and has a fine amorphous character.

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