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N Zimmermann

Publications and source records attributed to N Zimmermann.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1988

[Ultrahistochemical demonstration of blood group B substances on human erythrocytes with a lectin from 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.

ABO Blood-Group System↗
PubMed1987

Gadolinium and didymium (praseodymium/neodymium) cations as capture agents in lightmicroscopical histochemistry of acid and alkaline phosphatase.

In previous papers, cerium and lanthanum based methods for light-microscopical detection of acid and alkaline phosphatase activity were proposed. In this paper, the usefulness of other lanthanide cations such as gadolinium and praseodymium/neodymium cations as capture agents in phosphatase histochemistry is tested. It is evident that phosphate ions were sufficiently trapped by these cations. According to the lead and silver multistep procedures earlier described it is possible to visualize alkaline phosphatase activity in the brush borders of the intestine or kidney as well as acid phosphatase activity in the lysosomes. These methods can be recommended.

Acid Phosphatase↗
PubMed1987

Light microscopical localization of enzymes by means of cerium-based methods. V. Optimization of the cerium-lead (Ce-Pb)-technique for alkaline phosphatase.

A modification of the earlier published cerium-based technique for histochemical detection of alkaline phosphatase activity at light microscopical level (Halbhuber and Zimmermann 1985) is described. The reduction of the s-collidine concentration from 200 mmol to 50 mmol, increase of cerium ion concentration rom 1 mmol to 5 or 10 mmol, and sucrose concentration from 7.5% to 15% at increased from pH = 9.0 to 9.5 less than or equal to 9.9 in the incubation medium led to a high intensification of the histochemical reaction. The brush borders of the rat kidney (especially of the epithelial cells of the primary convoluted tubules) and of the enterocytes demonstrate black-brown tinged and precisely localized final reaction products. Moreover, a simplification of the histochemical procedure by employment of postfixed cryostat sections (small intestine) instead of the time consuming perfusion fixed material (kidney) is presented. Several fixatives were also tested. Nakane's periodate-lysine-paraformaldehyde (PLP) or the periodate-lysine-glutaraldehyde (GLP) fixations are superior to the classical glutaraldehyde/paraformaldehyde double fixation. The proposed optimized cerium-based techniques are recommended for a broad use.

Alkaline Phosphatase↗
PubMed1987

Preservation of red cell concentrates. Dependence on storage time of IgG binding, osmotic fragility, MCV, and surface area index.

In a comparative study of red cells from concentrates preserved in SAG medium without and with 30 mM sucrose, mannitol or sorbitol, resp., we determined the variation of IgG binding, osmotic fragility, MCV and the surface area index with storage time. IgG binding gave no conclusive results. Osmotic fragility turned out to be increased in simple SAG in comparison to the sugar-supplemented media. From measurements of microhematocrit and pH, the mean cellular volume (MCV), standardized to the initial pH value, turned out to decrease in all the media tested by not more than about 5 per cent after 3 weeks, and 10 per cent after 6 weeks. This is in advantageous contrast to the strong decrease in microhematocrit formerly observed in red cell concentrates in CDS-AG medium. Cells resuspended in simple SAG medium exhibited the smallest decrease in MCV. However, as inferred from data on hemolysis and vesiculation (D. Stibenz, accompanying paper), in these cells the loss of surface area proved to be maximal.

Blood Preservation↗
PubMed1987

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.

Erythrocyte Aging↗
PubMed1987

Morphometrical studies of erythrophagocytosis in the spleen.

The elimination of altered RBC was investigated morphometrically in the rat spleen by determination of the percentage of erythrophagosomes in macrophages. PHZ and diamide treated RBC led to a time dependent increase of the percentage of erythrophagosomes in comparison to normal spleens. The deformability of the target RBC was lowered as revealed by measurements with glass micropipettes. RBC-microvesicles and diamide treated RBC are primarily loaded with IgG and undergo a rapid elimination. PHZ-RBC and cells with a prolonged stay in the spleen are sequestrated by secondary mechanisms. Details of primary and secondary elimination are discussed.

Animals↗
PubMed1987

Membrane skeleton alteration--a factor promoting IgG receptor expression in the erythrocyte membrane.

IgG receptor expression after selective cross-linking of spectrin by means of diamide was investigated. A diamide concentration dependent IgG loading of erythrocytes was observed. Furthermore, diamide causes disturbance of lipid asymmetry, decrease of the anisotropy after topooptical staining of the glycocalyx, increase of the phagocytosis index and aggregation of the IMP's. Our findings support the hypothesis that the arrangement of the membrane skeleton at the inner aspect of the membrane is decisive not only for the lipid asymmetry but also for the spatial structure of the glycocalyx at the outer aspect of the membrane and thus for the degree of exposure and arrangement of IgG-receptors, which are thought to be localized at an extracellular portion of band 3 protein.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1987

Red blood cell aging--membrane skeleton alteration and IgG receptor expression.

Investigations were performed on aging of erythrocytes. It has been assumed that structural changes of the membrane result after exposer of the cells to certain environmental influences in vivo or in vitro. Cell aging can be connected with varying combinations of membrane structure disturbances. It is postulated that the messenger which signals membrane structure lesion is involved in a mechanism given by the expression of immunoglobulin G (IgG) receptor sites which bind autologous IgG1 and IgG3. This antibodies are cytophilic for macrophages. The performed studies demonstrated that an intact molecular arrangement of the membrane skeleton is not only a supposition for stabilization of the membrane asymmetry but also for IgG receptor masking to prevent an early elimination of the red blood cells from the organism.

Erythrocyte Aging↗
PubMed1986

Light microscopical localization of acid and alkaline phosphatase activity by lanthanum-lead-(La-Pb)-methods.

This paper reports on the application of lanthanum cations instead of cerium ions as captures to detect acid and alkaline phosphatase activity in the rat kidney and small intestine. The basis of the proposed histochemical reactions is a three step procedure in analogy to the earlier published cerium-based techniques: the lanthanum phosphate was converted into lead phosphate and finally into the brown coloured lead sulfide [lanthanum-lead-(La-Pb)-reaction]. The results reveal that the La-Pb-reactions are more sensitive in comparison to the Ce-Pb-reactions. Methodological experiments involving lanthanide specific blocking reactions are discussed. Lanthanum cations are recommended as a useful tool in histochemical capture techniques of phosphatases.

Acid Phosphatase↗
PubMed1986

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.

Acid Phosphatase↗
PubMed1986

Cytochemical and cellbiological investigations of the signal function of the erythrocyte plasmalemma--the membrane structure as code for cell life span.

The erythrocyte membrane was characterized with regard to its alteration of different degrees. A very important process of the membrane alteration is the expression of IgG receptor sites, which is connected with the binding of autologous IgG at the membrane. Several detection methods for membrane bound IgG were described: immunocytochemical techniques (Anti IgG--Anti Ferritin-sandwich technique, Anti IgG-Protein A-Gold- and the Silver enhancement technique), Antiglobulin agglutination test (Coombs test) and erythrocyte-macrophage test. Moreover, the topo-optical toluidine blue reaction and the ultra-histochemical NAD(H) oxidase reaction were checked to characterize the erythrocyte-macrophage interaction. Finally, the microvesiculation is understood as special form of the erythrocyte membrane disintegration, which is connected with a remodelling of an intact plasmalemma. The findings presented here summarize the fact that the membrane associated IgG regulates an immune-signal for the elimination of the erythrocytes in the Reticulo-Histiocytic-System in vivo and in vitro. Therefore, the structural integrity of the erythrocyte plasmalemma is the code for the cell life span.

Animals↗
PubMed1986

Topo-optical investigation of the erythrocyte membrane alteration after prolonged retention in the splenal reticulum.

The extent of the splenal transit time is an important factor which influences the immunological behaviour of the red blood cells. An increased transit time of the RBC in the splenal reticulum leads to conformational effects on the erythrocyte glycocalyx. They consist in spatial disorder of the oligosaccharide side chains of the glycophorins with their sialic acid residues. These spatial disorders of the sialic acid residues correspond with the expression of IgG receptors, which initiate the process of the secondary elimination of the red cells.

Animals↗
PubMed1986

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.

Animals↗