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K J Halbhuber

Publications and source records attributed to K J Halbhuber.

At least 37 records · Page 2Linked to original sources
PubMed1998

Laser scanning microscopy in enzyme histochemistry. Visualization of cerium-based and dab-based primary reaction products of phosphatases, oxidases and peroxidases by reflectance and transmission laser scanning microscopy.

The reflectance mode of confocal laser scanning microscopy is suitable to detect cerium-based primary reaction products of oxidases (CeIV-perhydroxide) and phosphatases (CeIII-hydroxy-phosphate converted into CeIV-perhydroxy-phosphate) as well as of DAB-based primary reaction products (Ni-DAB, Ni-FeII-DAB and CeIV-DAB complexes) of cytochrome C oxidase and peroxidases in vibratome, cryotome and semithin plastic sections. In combination with confocal detection 3D images with submicron spatial resolution can be obtained. Moreover, CeIV-perhydroxide, CeIV-perhydroxy-phosphate, CeIV-DAB complexes and catechol-DAB polymers are highly absorptive. Among other additives, especially stable nitroxyl radicals led to a distinct improvement of the DAB staining in terms of sensitivity and proper localization. This was proven in addition by means of blotting a horseradish peroxidase dilution series during several experiments. In sections it was easily possible to record reflectance signals and high transmission contrast at the wavelength of the exciting argon ion laser (preferentially 488 nm). The results of an imbibition study of cerium-containing model precipitates indicate that the cerium generally should be oxidized prior to observation because the index of refraction of CeIV compounds is considerably higher than that of the corresponding CeIII compounds. A comparative numerical assessment of reflection intensities from reflectant parts in morphologically similar sections is possible. Confocal laser scanning microscopy offers a unique way for high resolution detection of primary histochemical reaction products being sufficiently reflective and/or absorptive. The proposed techniques may open new methodological possibilities for basic research and for medical diagnosis.

3,3'-Diaminobenzidine↗
PubMed1996

Reflectance enzyme histochemistry (REH): visualization of cerium-based and DAB primary reaction products of phosphatases and oxidases in cryostat sections by confocal laser scanning microscopy.

In the present study the reflectance mode of confocal laser scanning microscopy was adapted to detect and to assess semiquantitatively cerium-based primary reaction products of oxidases [Ce(IV) perhydroxide] and phosphatases [Ce(III) hydroxyphosphate converted into Ce(IV) perhydroxyphosphate] as well as of the 3,3'-diaminobenzidine (DAB)-based primary reaction product of cytochrome c oxidase in cryostat sections. Confocal laser scanning microscopy offers a unique way of making visible histochemical reaction products which are weakly absorbant but sufficiently reflective. It was easily possible to record simultaneously the reflectance signals at the wavelength of the exciting laser (preferentially 488 nm) and the autofluorescence signals ( > 580 nm in our set-up) of glutaraldehyde-fixed tissue. The results of an imbibition study of cerium-containing model precipitates indicate that the cerium, generally, should be oxidized prior to observation because the index of refraction of Ce(IV) compounds is considerably higher than that of the corresponding Ce(III) compounds. An attempt at comparative numerical assessment of reflection intensities from reflectant parts in morphologically similar sections is presented. The proposed technique may open new possibilities in enzyme- and immunohistochemistry.

3,3'-Diaminobenzidine↗
PubMed1996

Quantitative assessment of primary cerium reaction product formed by glucose-6-phosphatase activity in a male rat liver: an image analysis study.

Glucose-6-phosphatase (G6Pase) activity has been determined in periportal and pericentral areas of the liver of normal male rats. Measurements were performed on unfixed cryostat sections mounted on semipermeable membranes. In the present study, the oxidized primary reaction product of a cerium-based histochemical method [Ce(IV)perhydroxyphosphate] instead of the final reaction product after a second-step incubation was measured. For quantification of the amount of Ce(IV)perhydroxyphosphate formed the digital image analyzing system Quantimet 500+ was used. Estimated values of optical densities of Ce(IV)perhydroxyphosphate over test areas were employed for calculation of kinetic parameters of (G6Pase). Highest activities of G6Pase (higher Km and Vmax levels) were found in periportal areas of the rat liver, indicating a higher amount of active enzyme molecules and a lower affinity for the substrate. Differences in values for both Km and Vmax between periportal and pericentral zones were highly significant and closely comparable to those for male fed rats. Correlations between Km and Vmax were significant for periportal as well for pericentral liver areas. The results of the present study thus allow the same biological implications as histochemical methods employing a final reaction for quantification of enzyme activities. The present method avoids the drawbacks of enhancement reactions and demonstrates the feasibility of in situ analysis of enzyme kinetic parameters by quantification of oxidized primary cerium reaction products.

Animals↗
PubMed1996

3D resolved two-photon fluorescence microscopy of living cells using a modified confocal laser scanning microscope.

Non-linear 3D imaging of fluorophore-labelled vital cells has been performed by femtosecond near infrared (NIR) microscopy. Ultraviolet and visible transitions of intracellular fluorophores, such as Fura-2, Calcium Green, Rhodamine 123 and fluorescent microspheres, were excited via simultaneous absorption of two 780 nm photons provided by a tunable Ti:Sapphire laser. The femtosecond laser was coupled to a conventional upright Zeiss confocal laser scanning microscope expanding its one-photon capabilities to 3D resolved two-photon microscopy. Pinhole-free non-linear 3D imaging was possible with 400 nm lateral and approximately 1 micron axial resolution. Axial resolution could be further improved by using an additional detection pinhole. The NIR average power and pulse width at the sample were adjusted to be 1-4 m W and 150-200 fs, respectively. Higher power levels resulted in cell damage as demonstrated by photoinduced lysis of human erythrocytes. The powerful capabilities of this universal microscope were demonstrated by 3D imaging of two-photon excited fluorophore-labelled macrophages during phagocytosis of fluorescent microsized beads.

Erythrocytes↗
PubMed1995

Light and electron microscopical demonstration of the ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity (K-NPPase) using a Ce-Mg-double capture technique.

The cerium-based method of Kobayashi et al. for the histochemical demonstration of K-NPPase activity was improved. Besides Ce3+ additionally Mg2+ ions as orthophosphate capture were employed (double capture technique). For light microscopical purposes the Mg-phosphate was converted into Ce-phosphate by treatment of the sections with Ce-citrate yielding higher quantity of reaction product. Unspecific background staining was eliminated by EGTA. In the electron microscope this technique brought about fine granular reaction products without diffusion artefacts.

4-Nitrophenylphosphatase↗
PubMed1994

Is the brush border membrane of the intestinal mucosa a generator of "chymosomes"?

1. The microvilli of enterocytes in calf intestine demonstrate high levels of vesiculation activity at the top and at the basal region. 2. The morphology of the vesicles associated with microvilli (100-500 nm diameter, unilamellar, few intramembraneous particles, high AP activity) is very similar to the morphology of vesicles found in the chyme. 3. Vesicles can be purified 6-10 fold from chyme of the calf intestine applying a Mg(++)-precipitation method, used for brush border membrane preparation. 4. Specific activities of alkaline phosphatase and disaccharidases were found to be much higher in chyme vesicles than in the mucosa. 5. Phospholipid content and phospholipid composition is in chyme vesicles different from brush border membrane vesicles. 6. The characterized chyme vesicles are referred to as chymosomes. We consider the mucosa as a large-scale generator of chymosomes, i.e. digestive enzymes bearing vesicles.

Alkaline Phosphatase↗
PubMed1994

Dysmorphic erythrocytes in glomerulonephritis. 1. Electron microscopical and histochemical investigation.

Dysmorphic erythrocyte malformation in urine is characteristic of glomerulonephritis. The mechanisms leading to this phenomenon are still unknown. To obtain evidence of the site as well as of the process of erythrocyte damage, electron microscopical and histochemical investigations of renal biopsy materials from 19 patients with histologically defined glomerulonephritis were performed. The results suggest that the initial damage of erythrocytes in the glomerular area is reasoned by enzymatic glycocalyx destruction. On passage through the tubular system the osmotically sensitized surface altered cells undergo rapid hemolysis and losses of membrane skeletal proteins leading to dysmorphic shape transformations.

Adult↗
PubMed1994

Dysmorphic red cell formation in glomerulonephritis. 2. In vitro generation of dysmorphic erythrocytes.

In the present study influences leading to in vitro formation of dysmorphic erythrocytes were studied. Fresh and surface altered erythrocytes pretreated with several proteases were passed through different solutions representing distinct nephronís fluids. After passage of intact erythrocytes only 12-15% dysmorphic cells were observed. In case of protease treated cells the number of dysmorphic cells rose to 35-80%. The hemoglobin content was decreased. PAGE and electron microscopical findings demonstrated substantial losses of transmembrane and membrane skeleton proteins. It is assumed that surface protein degradation, loss of membrane skeleton proteins and hemolysis seem to be closely associated with dysmorphic malformation of urinary erythrocytes characteristical for glomerulonephritis.

Anisotropy↗
PubMed1993

Evaluation of experimentally induced enzymatic alterations on the erythrocyte membrane by use of alcian blue.

The binding of the polycationic dye alcian blue to erythrocytes is investigated at pH 6 and pH 3 after treatment with neuraminidase and proteolytic enzymes. The portion of the dye bound by membrane-negative charge-carriers, as well as by hydrophobic interactions with mainly membrane protein compounds, is calculated. It is demonstrated that not only the sialic acid, as the main negative charge domain, but also proteins are interacting with the dyestuff. Proteolytically split membrane fragments contain a considerable amount of alcian blut that cannot be associated with the estimated sialic acid content.

Alcian Blue↗
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↗