Herwart Feuerstein

The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure

SummaryNew 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°–60° 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.

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 cytochromec 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.

[The altered membrane of the erythrocyte. I. Ultrahistochemical and biocellular investigations for the detection of activated acetylcholinesterase (ACHE) and demasking of IgG receptor sites (author's transl)].

Zusammenfassung Es wurde versucht, verschiedene Zustandsformen der Erythrocytenmembran im Hinblick auf den Grad ihrer Desintegration naher zu charakterisieren. Als Kriterien wurden die Bindungsfahigkeit gegenuber autologem bzw. allogenem IgG, der Aktivierungsgrad der am intakten Erythrocytenplasmalemm inhibierten membransessilen Acetylcholinesterase und die Mernbranvesiculation herangezogen. Die Befunde belegen, das offenbar mit dem Schweregrad der Membrangefugestorung die IgG-Bindung anwachst und die Acetylcholinesterase zunehmend aktiviert wird. Das Enzym kann dann spektrophotometrisch und ultrahistochemisch erfast werden. Die Mikrovesiculation wird als Ausdruck tiefgreifender Membrangefugestorungen auigefast. Am konservierten Erythrocyten wird sie als ein lokaler Membran-Remodellierungsprozes angesehen, Starkere Erythrocytenschadigung, wie nach mechanischem Stress, Warmesohock und Inkubation in Harnstoff, fuhrt zu vergleichsweise hoheren Vesiculationsraten, teilweise zu Zellfragmentation. An stark spektrinverarmten Erythrocytenschatten tritt Mikrovesiculation spontan ein und fuhrt zum volligen mikrovesicularen Zerfall der Schattenmembran. Das membranassoziierte autologe IgG wird mit immunologischen und ultrahistochemischen Methoden nachgewiesen. Seine Bedeutung als homoostatisch wirksames Immunsignal fur die Elimination von in vivo oder in vitro gealterten Erythrocyten, Erythrocytenschatten und -vesiceln durch das Reticulo-Histiocytare-System wird anhand von Modellversuchen aufgezeigt. Molekulare Mechanismen zur IgG-Receptordemaskierung und A.cetylcholinesteraseaktivierung in der alterierten Erythrocytenmembran werden diskutiert.

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.

Immunologische und enzymatische Aktivität nach Fixierung mit Diimidoester Verbindungen

Zusammenfassung In der vorliegenden Arbeit wird uber die Fixierungseigenschaften zweier Diimidoester fur histoehemische Untersuchungen berichtet. Es wird gezeigt, das nach diimidoesterinduzierten Vernetzungsroaktionen an RBC die Receptoraktivitat dieser Zellen weitgehend erhalten bleibt. Diese Eigenschaften machen es moglich, amidinierte RBC als Immunadsorbens fur Hamagglutinations-reaktionen einzusetzen. An peroxidasestimulierten Plasmazellen wird beobachtet, das die Reaktivitat intracellularer Antiperoxidase-Antikorper nach Amidiniorung ebenfalls sehr hoch ist. Am Beispiel der histochemischen Nachweisreaktionen fur SDH, alkalische bzw. saure Phosphatase und unspezifische Esterase in amidinierten Gewebsschnitten wird die Uberlegenheit der Diimidoesterfixierung gegenuber der konventionellen Aldehydfixation verdeutlicht. Die fur licht- und elektronenmikroskopisch-histochemische Nachweisverfahren erforderlichen Amidinierungsbedingungen werden vorgestellt.

Light microscopical localization of enzymes by means of cerium-based methods. IV. 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.

Alteration of the enucleating erythroblast glycocalyx

Plasmalemmal differentiation of the enucleating normoblast of rabbit and rat was studied by means of cytochemical methods and freeze-etching. Staining with colloidal iron revealed about identical amounts of iron particles bound to various areas of the normoblast membrane. Cationized ferritin and ruthenium red, likewise, failed in the demonstration of significant changes of the enucleating normoblast glycocalyx. Despite these findings the topo-optical staining with toluidine blue showed the plasmalemmal envelope of the protruding normoblast nucleus moderately birefringent, clearly discriminated from the intense anisotropic staining of the future reticulocyte membrane. The ferritin-labeled snail lectin anti AHP localized a great number of binding sites at the plasmalemmal envelope of the nucleus under extrusion. That is in sharp contrast with rather low lectin binding to the future reticulocyte membrane which amounts to about 30 to 50% of the nuclear envelope label. The findings provide evidence of unmasking of bindings sites of the normoblast membrane. Apparently, the effect is due to conformational changes of the cell membrane, rather than it could be attributed to degradation of glycoproteins. Moreover, enucleation kinetics may also be related to supramolecular changes of membrane structure albeit missing evidence for the rearrangement of membrane particles.

Spatial arrangement of some erythrocyte membrane receptor sites.

Alcian Blue proved to be a hemagglutinin specific for negatively charged receptors. Either sialylization or tryptic degradation of glycoproteins of human erythrocytes greatly diminished the agglutinability by Alcian Blue. Studies with group A antiserum, anti-AHP and the lectin of Lens culinaris demonstrated the masking efficiency of erythrocyte major glycoproteins. Previous proteolytic digestion enhanced the agglutinability of human red blood cells due to uncovering of what is considered glycolipidic group A receptors. The findings provide indirect evidence for the spatial arrangement of erythrocyte receptor sites.

Ultrahistochemischer Nachweis der Blutgruppensubstanz B menschlicher Erythrocyten mit einem Lectin aus Salmo gairdneriRich.

Summary 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.

A simplified procedure for estimation of lipid phosphorus in erythrocyte membranes

For the estimation of phospholipids and the other components of the erythrocyte membrane, methods are desirable that supply reproducible results, are simple and time-saving and, thus, practicable in a routine laboratory. Nowadays, nearly all procedures used for estimation of lipid phosphorus in the erythrocyte membrane require the application of extraction methods. The results of a variety of modifications of these methods are strongly affected by the composition of the solvent mixture as shown by Ways and Hanahan [l]. In addition to the extraction procedures of Folch et al [2], those of Rose and Oklander [3] have been applied in many investigations. However, the relatively considerable methodical deficiencies of all extraction procedures should not be ignored. To remove the disturbing non-covalently bound, phosphorus-containing compounds from the lipid extracts, a variety of wash procedures and specific after-treatments are necessary. Moreover, most of the methods known demand a quantitative preparation of erythrocyte ghosts before extraction. The quantitative manipulation of the ghosts during washing is difficult and high speed centrifugation (12000 X g) is necessary for the avoidance of losses. In order to overcome these difficulties, we have tested the polycationic dye Alcian blue for a quantitative preparation of erythrocyte ghosts. Some years ago this compound was used by us for haemagglutination experiments [4]. In the following, we will discuss the application of the erythrocytedye aggregates thus formed in a reproducible assay for the lipid phosphorus of the red cell membrane. This was compared with a method using an extraction procedure of a normal red cell suspension. In addition, some individual phospholipid classes were estimated by an extraction procedure of the Alcian blue ghost aggregates and also compared with our current method.