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.

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.

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.

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.

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).

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.

[How thick is the glycocalyx of human erythrocytes?]. / Wie dick ist die Glykokalyx des menschlichen Erythrocyten?

The aim of the experiment was to determine the thickness of the glycocalyx of human erythrocytes of the blood group A. For that purpose, the distance between the electron dense contrasted lipid layer of the plasmalemma and gold sol particles loaded with Helix pomatia lectin was determined. The mean thickness of the glycocalyx under this conditions was 5.9 nm.

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.

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.

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.

[Alcian blue: an agent for quantitative production of erythrocyte preparations and for understanding erythrocyte changes during aging]. / Alcianblau--ein Agens zur quantitativen Gewinnung von Erythrozyten-Präparationen und zur Erfassung von Altersveränderungen am Erythrozyten.

The polycationic dye Alcian blue 8 GS fits for a simple quantitative preparation of erythrocyte membrane as well as a tool for judgement of erythrocyte membrane alterations in blood preserves in different age of stage.

[Ultrahistochemical demonstration of blood group B substances on human erythrocytes with a lectin from Salmo gairdneri RICH]. / Ultrahistochemischer Nachweis der Blutgruppensubstanz B menschlicher Erythrocyten mit einem Lectin aus Salmo gairdneri RICH.

From the roe of Salmo gairdneri RICH., a lectin was isolated which agglutinates specifically human erythrocytes of blood group B. For cytochemical labelling of the blood group substance B on the surface of human erythrocytes, an indirect approach was chosen. By means of a polyclonal antibody from the rabbit against the B-specific fish lectin, electronmicroscopic presentation was performed with Protein A gold using a multistep method. For quantification gold labelling was partially followed by a silver technique.

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.

Detection of IgG bound at the erythrocyte membrane by means of an immunohistochemical gold-silver technique.

The binding of IgG at the erythrocyte membrane during aging plays an important role in the elimination process of the cells by the reticulohistiocytic system. By means of an indirect protein A gold- and protein A gold-silver-method bound IgG was detected immunocytochemically on physiologically aged, pronase and neuraminidase treated red blood cells (RBC). At the light and electron microscopic level an increased binding of IgG at "old" as well as enzymatic treated cells in comparison to "young" and normal RBC was noted. The silver enhancement of gold particles visualized the immunostaining and permitted the semiquantitative analysis of the RBC by a scanning microdensitometer.

Light microscopical localization of enzymes by means of cerium-based methods. I.V. Optimization procedures for acid phosphatase.

The earlier described cerium based histochemical reaction for acid phosphatase [Ce-Pb-reaction, Zimmermann and Halbhuber (1985)] was optimized. The target tissues (kidney, intestine) were fixed by perfusion with glutaraldehyde in cacodylate or piperazine buffer in anesthetized animals. Postfixation of prefixed sections is not advantageous because of the detectable repressing of the enzyme activity. Moreover, the employment of unfixed cryostat sections, which were postfixed, was always connected with a complete abolition of the acid phosphatase activity. The optimal concentration of the primary capture cerium III chloride in the incubation medium is about 1 mmol. Lower concentrations lead to an incomplete histochemical detection of phosphatase activity in lysosomes. The treatment of cryostat sections of perfusion fixed tissue with borohydride (diminution of aldehyde induced cross links) or with dimethylsulfoxide (extraction of lysosomal materials or the well known vehicle property) brought about an improvement of the penetration capacity for cerium-III-cations into the target structures. After conversion of the cerium phosphate (primary specific reaction product) into cerium perhydroxide, oxalate or fluoride, the Ce-Pb-reaction was negative. Therefore, these blocking reactions represent specific inhibition controls, which indicates the formation and presence of cerium phosphate. On the basis of these reactions it is possible to check the specificity of the histochemical Ce-Pb-reaction for phosphatase activity in sections.

Immunocytochemical investigations of the membrane of experimentally altered and physiologically aged erythrocytes.

The expression of IgG receptor sites during aging of red blood cells plays an important role in the elimination process of these cells by the Reticulo Histiocytic System. By means of an indirect protein A gold-method, membrane bound IgG was detected immunocytochemically on pronase and neuraminidase treated red blood cells as well as on physiologically aged red cells. The silver enhancement of the gold particles led to an improved labeling of the bound IgG favouring its quantification at both electron and light microscopic level. The result of this gold-silver-technique was analyzed semiquantitatively at the light microscopic level by use of a Vickers scanning microdensitometer. It was shown that the aging of erythrocytes as well as the enzymatic alteration of the erythrocyte glycocalyx by pronase and neuraminidase are accompanied with an unmasking of IgG receptor sites followed by an IgG loading of the cells in presence of autologous serum or plasma. The experimental results, which are presented here indicate that a silver enhancement of gold particles is sufficient to signalize changes in membrane bound ligands at low concentrations.

Light microscopical localization of enzymes by means of cerium-based methods. III. Visualization techniques for cerium phosphate.

Cerium-based methods are more and more used for the electron microscopic localization of phosphohydrolases. By means of the earlier described Ce-Pb-technique, it is possible to localize these enzymes on the light microscopical level. The final product of this reaction is lead sulfide. In addition to this technique, other visualization methods for the light microscopically not visible cerium phosphate are proposed. 3 successful techniques are described in the report: The cerium perhydroxide reaction. By means of H2O2 cerium phosphate is converted into cerium perhydroxide which has an orange-yellowish colour. The manganese dioxide reaction with the conversion of cerium phosphate into cerium oxalate, which is able to reduce permanganate into the hardly soluble brown coloured manganese dioxide. A silver technique (Ce-Pb-AgS-method), which is characterized by the conversion of cerium phosphate into lead phosphate and in a second step to lead sulfide. At the active sites of the lead sulfide, the reduction of silver ions takes place. The reduced silver is converted in a final step into silver sulfide. The enzyme activity is represented by a brown or black coloured staining.