Search PubMed⌕ Search

Biomedical subjects

K J Halbhuber

Publications and source records attributed to K J Halbhuber.

At least 19 recordsLinked to original sources
PubMed1992

Modified cerium-based and Gomori-based cerium methods for light microscopic phosphatase histochemistry: the cerium-perhydroxide-diaminobenzidine-nickel (Ce-H2O2-DAB-Ni and Ce/Ce-H2O2-DAB-Ni) two-step procedures.

Some modified cerium-based and Gomori-based cerium methods for the demonstration of phosphatase activity in cryostat sections were described. Dextrane as stabilizing agent was added to the incubation media for ATPase, 5'-Nase, and TPPase. The oxidation of the CeIII-phosphate primary reaction product in a separate step by H2O2 before the DAB incubation yielded an increase of the intensity of the DAB-based visualization reaction (Ce-H2O2-DAB-Ni two step method). The sensitivity of the histochemical enzyme reaction was remarkably increased if CeIII-ions were employed as amplifying agent (Ce/Ce-H2O2-DAB-Ni two-step method). A new suitable DAB medium consisting of 0.015% DAB, 2.0% Ni-sulphate, 15% methanol, and 0.005% H2O2 in 0.1 mol/l acetate buffer, pH = 5.2, was used. The disadvantage of diffuse background staining has been overcome by addition of 15% methanol to the DAB solution. Electrovalently bound CeIII (cerophilia) was removed by treatment of the incubated sections with CeIII-citrate (CeIII-complexation). In addition, a novel membrane floating incubation for sections is proposed. At present, the modified procedures are some of the most sensitive modes for the demonstration of phosphatases and improve the earlier described cerium-DAB one-step technique (Halbhuber et al. 1988b).

Animals↗
PubMed1992

Light microscopical demonstration of non-specific alkaline phosphatase activity with an incubation medium containing cerium and two calcium as the capturing agents. The cerium/calcium-hydrogen peroxide-P-phenylenediamine/pyrocatechol (Ce/Ca-H2O2-PPD/PC) double capture technique.

A new method for the light microscopical demonstration of alPase activity in cryotome sections by using simultaneously cerium and calcium as capturing agents (double capture technique) is described. This method has an increased sensitivity compared with the single cerium-based and the Gomori based-cerium (single calcium and cerium converted) with techniques described previously. Presuming that the enzymatic activity during incubation of sections in the presence of a defined capturing agent is constant, the increased sensitivity after employment of the double capture method could be attributed to a decrease of enzyme inhibition by cerium through the presence of calcium. Based on model experiments it is assumed that calcium phosphate and cerium phosphate are the primary reaction products, the former converting into cerium phosphate already during incubation. The remaining calcium phosphate is converted completely by treatment with cerium citrate solution (conversion reaction). After oxidation with H2O2 the cerium perhydroxyphosphate was visualized in a paraphenylenediamine/pyrocatechol (Hanker-Yates reagent) solution without H2O2 to give a black reaction product. This visualization procedure is superior to the DAB or DAB-Ni mode as published earlier. Some results concerning the mode of inhibition of the pseudoperoxidase activity of the hemoglobin are presented.

Alkaline Phosphatase↗
PubMed1991

Employment of merocyanine 540 fluorescence to form diaminobenzidine (DAB) oxidation product: a photoconversion method for the visualization of erythrocyte membrane fluidity for light and electron microscopy.

Intact native red blood cells (RBC) and treated RBC preparations were labelled with MC 540 and irradiated in the presence of diaminobenzidine (DAB). The polymerized diaminobenzidine reaction product is permanently stable in comparison with the labile fluorescence labelling. The brownish stained DAB polymerization product (DAB brown) and osmium black (after conversion of DAB brown with OsO4) allow the densitometrical determination with the light microscope. The latter product can be directly observed in the electron microscope. A direct correlation exists between the fluorescence intensity and the polymerized diaminobenzidine staining. It can be deduced that the enhancement of the DAB mediated contrast is reflecting an increased fluidity of the red cell membrane. The reaction was successful with all red cell preparations tested. This method is also suitable for the determination of fluidity changes in other cell membranes.

Diamide↗
PubMed1991

The perinatal development of glucose-6-phosphatase activity distribution pattern in rat liver. A microdensitometrical study.

The ontogenetic development of the intralobular distribution pattern of glucose-6-phosphatase activity in the rat liver is described in terms of histochemical changes determined with microdensitometry. A newly developed cerium-lead technique was employed and compared with the common lead technique optimized by Teutsch (1978a). The cerium technique has advantages, meets the prerequisites for quantitative determinations and yields results comparable to biochemically obtained data from microdissected tissue. The first signs of a heterogeneous distribution pattern of glucose-6-phosphatase activity are observed on the 3rd d after birth, and differences between periportal and centrolobular areas are largest around 10th and 15th d. At 30th d after birth, the adult pattern is complete with a centrolobular glucose-6-phosphatase activity of 67% of the periportal value.

Aging↗
PubMed1991

Improved light microscopic demonstration of D-amino acid oxidase activity in cryotome sections using cerium ions as capturing and amplifying agent--the Ce/Ce-H2O2-DAB procedure.

The light microscopical demonstration of D-amino acid oxidase (AAOX) activity with cerium (Ce III) as the capturing agent was improved. The incubation medium was stabilized by the employment of triethanolamine and detrane complexed cerium. A considerable increase in intensity of the reaction was accomplished by treatment of the AAOX-incubated sections with Ce III which reacted with the primary reaction product Ce IV-perhydroxide to form Ce IV-hydroxide. In this way the primary reaction product was reduced and enlarged concomitantly. The Ce IV-hydroxide was converted into Ce IV-perhydroxide by H2O2, which was visualized by blue-black stained Ni-DAB complexes. Thus, Ce III is used as capturing agent as well as amplifier (Ce/Ce-H2O2-DAB method). The primary reaction product Ce III-phosphate formed by coreacting phosphatases was selectively extracted by citrate containing glycine-NaOH buffer while Ce IV-perhydroxide remained in the sections. In model experiments it was proven that the perhydroxide groups in the Ce IV-perhydroxide compound initiate predominantly the DAB polymerization while the contribution of Ce III and Ce IV is small.

3,3'-Diaminobenzidine↗
PubMed1991

A new visualization principle of cerium phosphate reaction product of phosphatases in cryotome sections for light microscopy--the cerium-oxalate-osmium-silver (Ce-Ox-Os-Ag) method.

A new visualization principle for the detection of cerium phosphate reaction product of phosphatases in light microscopy is described. The new mode is based on the conversion of cerium phosphate into cerium oxalate. The latter is able to react with OsO4. In this way osmium black is formed, staining enzymatic activity sites grey or greyish-black. Utilizing the argyrophilia of osmium black, the staining contrast could be remarkably intensified by posttreatment with Ag-proteinate (Ce-Ox-Os-Ag procedure). This procedure is of similar sensitiveness in comparison to the DAB-Ni method, proposed earlier. The advantages are a very pale background staining and a complete suppression of the cerophilia. Moreover, it substitutes to DAB, a probable potent carcinogen. The method is time-saving when the processing of the reactions was stimulated in a microwave oven.

Animals↗
PubMed1991

Cerium as amplifying agent--an improved cerium-perhydroxide-DAB-nickel (Ce/Ce-H2O2-DAB-Ni) method for the visualization of cerium phosphate in resin sections.

A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in 0.5 - 2.0 microns semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with OsO4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique.

3,3'-Diaminobenzidine↗
PubMed1990

The binding of a polyclonal antibody against human band 3 to in vitro aged erythrocytes.

In vitro aged human erythrocytes were checked for their anti-band-3-antibody loading by means of a protein A-gold technique. The in vitro-ageing procedures are introduced as an alternative to the questionable preparation of in vivo aged cells. They include the influences of NaF in various concentrations, of heat (49 degrees C) and urea. After the treatment with 20 mmol/l NaF and more, the IgG loading increases significantly. In contrast to that, however, exovesiculation induced by heat and urea lower the IgG loading. Possible mechanisms for the growing accessibility of the epitopes concerned are discussed in connection with changes occurring during the physiological ageing processes.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1990

Polarization-optical investigation (topo-optical analysis) of the structure of the human erythrocyte glycocalyx. Influence of pH, ionic strength and diamide-induced spectrin cross linking.

The value of membrane anisotropy after fixation and topo-optical analysis of erythrocytes stained with toluidine blue is a measure for the degree of spatial order of the dyestuff-binding acidic residues of the glycocalyx and thus a parameter for the characterization of the glycocalyx structure. Lowering the pH value and/or the ionic strength of the staining medium results in a decrease of membrane anisotropy indicating a lower order of the anionic residues. In agreement with the findings of other authors this phenomenon seems to be connected with an expansion of the glycocalyx. Diamide-induced oxidative crosslinking of spectrin before fixation and staining with toluidine blue at physiological pH and ionic strength also results in a decreased anisotropy. This indirect influence on the glycocalyx structure may be caused by an increase of the charge density within the glycocalyx due to a diamide-induced rearrangement of the membrane skeleton and of the transmembrane proteins bound to it.

Diamide↗
PubMed1990

[Does induced Heinz body formation lead to the liberation of IgG receptors on the erythrocyte membrane?].

Immunofluorescence microscopical and biochemical studies led other authors to the conclusion that the formation of membrane-bound Heinz bodies at these parts of the plasmalemma of erythrocytes leads to clustering of band 3-protein and increased binding of IgG. We failed to detect immunocytochemically an increased IgG binding over phenylhydrazine induced membrane-bound Heinz bodies in otherwise intact erythrocytes using transmission electronmicroscopy and we also did not notice any clustering of intramembraneous particles over the numerous Heinz bodies by means of freeze etching. Especially at higher phenylhydrazine concentrations the erythrocytes show formation of vesicles and hemolysis accompanied by increased IgG binding and clustering of the intramembraneous particles. Photographs of such cells obtained by immunofluorescence microscopy are very similar to the pictures known from the literature.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1989

Topo-optical studies of gradually disintegrated erythrocyte membrane derivates: different kinds of ghosts.

Several kinds of ghosts from human erythrocytes (blood-group A1D) were investigated by the topo-optical Toluidine-Blue (TB) reaction. In comparison to intact cells, all ghosts demonstrated a decreased TB-anisotropy. These results reflect an altered glycocalyx structure of ghosts, especially conformational changes of the TB-binding N-terminal extracellular segments of the glycophorines. It was assumed that this structural glycocalyx alteration was caused by substantial losses of membrane skeleton components during the ghost preparation. Moreover, disturbed molecular interactions between the membrane skeleton and the glycocalyx may contribute to this effect. Therefore, the glycocalyx and the membrane structure of ghosts in general are significantly different from the membrane of the intact erythrocyte. The experiments show that the effects of the reconstitution procedures for ghost membranes are restricted to a reconstitution of a defined membrane function (i.e. dynamic barrier for monovalent cations) in a widely disturbed membrane structure.

Acetylcholinesterase↗
PubMed1989

[The elimination mechanism of erythrocytes in vivo].

The erythrocytes exhibit an unusual cyto-architecture. Because the cytosol is without organelles, the erythrocytes are cells, which have got only one single organelle, i.e. the plasmalemma. It reacts extremely sensitively to environmental factors with changes of the membrane structures, especially with an expression of IgG-and lectin receptor sites. This takes place during in vivo ageing, after attack of hydrolytic enzymes and after alteration of the membrane skeleton. The membrane bound IgG mediates the endocytosis of the macrophages (primary elimination). The role of the lectin receptors is unclear. The cell trapping (secondary elimination) is restricted to cells with extremely decreased deformability.

Antigens, Differentiation↗
PubMed1988

Light-microscopic histochemistry of non-specific alkaline phosphatase using lanthanide-citrate complexes.

New lanthanide methods for the histochemical detection of non-specific alkaline phosphatase in the light microscope are described and compared with already existing techniques for the light microscopical demonstration of this enzyme. To avoid formation of insoluble lanthanide hydroxide at alkaline pH citrate complexes with the capture ions cerium, lanthanum and didymium were used. A molar ratio of 11 mM citrate/14 mM capture reagent is proposed. For preincubated sections, pretreatment in chloroform-acetone and fixation in glutaraldehyde, for non-preincubated sections fixation in glutaraldehyde yielded the best results. 4-Methylumbelliferyl and 5-Br-4-Cl-3-indoxyl phosphate were found to be the most suitable substrates. For routine purposes 4-nitrophenyl, 1-naphthyl, 2-naphthyl and 2-glycerophosphate were also sufficient; naphthol AS phosphates were inferior but still suitable. After incubation for 5-60 min at 37 degrees C lanthanide phosphate was converted into lead phosphate which was visualized as lead sulfide. At pH 9.2-9.5 enzyme activity was demonstrated at many sites such as intestinal, uterine, placental, renal and epididymal microvillous zones, plasma membranes of arterial, sinus and capillary endothelial cells, vaginal and urethral epithelium, smooth muscle cells, myoepithelial cells as well as excretory duct cells of salivary and lacrimal glands and in secretory granules of laryngeal glands. In comparison with Gomori's calcium, Mayahara's lead, Burstone's and Pearse's azo-coupling, McGadey's tetrazolium salt and Gossrau's azoindoxyl coupling technique the lanthanide methods detected alkaline phosphatase activities at identical or additional sites depending on the respective procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1988

The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure. New methods for light microscopic phosphatase histochemistry and immunohistochemistry.

New light microscopic visualization methods were developed for the histochemical detection of non-specific alkaline and acid phosphatase, Mg-, Ca- and Na, K-dependent adenosine triphosphatase, myosin adenosine triphosphatase, glucose-6-phosphatase, 5'-nucleotidase and thiamine pyrophosphatase with cerium ions as trapping agents in cryostat and plastic sections. The techniques are based on the conversion of cerium phosphate into cerium perhydroxide by H2O2 which decomposes at 55 degrees-60 degrees C into cerium hydroxide and oxygen radicals. These radicals are able to oxidize diaminobenzidine (DAB) to DAB brown. Addition of nickel ions to the DAB-H2O2 mixture generates bluish-black stained nickel-DAB complexes. Compared with the classical metal precipitation, azo, azoindoxyl and tetrazolium procedures the H2O2-DAB and especially the H2O2-DAB-nickel methods provided identical or superior results in catalytic phosphatase histochemistry and immunohistochemistry when using non-specific alkaline phosphatase as the enzyme label.

3,3'-Diaminobenzidine↗
PubMed1988

New, improved lanthanide-based methods for the ultrastructural localization of acid and alkaline phosphatase activity.

New, improved techniques for the ultrastructural localization of acid and alkaline phosphatase activity using lanthanide cations as the trapping agent were developed. Delayed penetration of the capture ions and the incubation constituents into cellular compartments was prevented by pretreating specimens with borohydride/saponin. Both the concentration of the capture agent in the incubation medium and the incubation time of the tissue specimens were optimized to achieve a satisfactory cytochemical reaction and to avoid precipitation artefacts caused by local matrix effects. The conversion of cerium phosphate into the almost insoluble cerium fluoride minimized losses of the reaction product during postincubation processing. Moreover, lanthanum itself as well as lanthanides other than cerium, e.g., gadolinium and didymium (praseodymium, neodymium), were successfully applied and can be recommended as capture agents for phosphatase cytochemistry.

Acid Phosphatase↗
PubMed1987

Topo-optical investigations of human erythrocyte glycocalyx conformational changes induced by dextran.

Cell surface properties are involved in the aggregation process of red blood cells. Using the topo-optical toluidine blue reaction, conformational changes of the glycocalyx (main component glycophorin A) were found when red blood cells were incubated and fixed in the presence of dextran. Relative differences in optical path as a measure of red blood cell membrane anisotropy decreased in relation to dextran concentration during fixation. These conformational changes could not be detected by electrophoretic measurements. When incubating, fixing and staining red blood cells in the presence of dextran, anisotropy decreased only at low dextran concentrations and increased at rising dextran concentrations. This biphasic course of differences in optical path seems to be due to different effects of dextran superimposing upon each other: a disturbing influence on the spatial order of sialic acid carrying oligosaccharide side chains due to H-bond interaction, and an increase in the size of dye aggregates and suppression of the thermal motion of macromolecules at higher dextran concentrations.

Dextrans↗