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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The activity of nonspecific alkaline phosphatase (E.C. 3.1.3.1) in developing teeth and bone of human fetuses and young macaque monkeys has been studied by means of histochemistry. The incubations for alkaline phosphatase were performed at pH 8.2 using naphthol-AS-MX-phosphate as substrate and Fast Blue RR salt or Fast Red Violet LB salt as couplers. By means of pretreatment with heat (56 degrees C), or addition of sodium metavanadate, ortho- or pyrophosphate, two alkaline phosphatases were demonstrated in the developing teeth. Prior to hard tissue formation all alkaline phosphatase activity was inhibited by the addition of vanadate, phosphate, or by pretreatment with heat. Pretreatment with heat or addition of vanadate or phosphate also inhibited alkaline phosphatase activity in the odontoblasts and in the pulpal connective tissue, whereas the activity in the subodontoblastic cell layer, stratum intermedium, outer enamel epithelium, and the the outer cells of the reduced enamel epithelium were much less affected. A week resistant activity was also noted in odontoblasts and pulpal connective tissue.
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PMID:Alkaline phosphatase in developing teeth and bone of man and macaque monkey. 9 18

Monkey pulps were homogenized in a Triton tris solution. After three centrifugation steps (800, 20000, and 105000 g) the supernatant was applied on acryl amide columns at pH 7.5 in a tris-diethyl barbituric acid buffer. Electrophoresis was performed at a constant current of 2.5 mA per gel column at 18--20 degrees C. Incubations for alkaline phosphatase (E.C. 3.1.3.1) were carried out at pH 8.3 using naphthol-AS-MX-phosphate as substrate and Fast Red Violet LB salt as coupler. Incubations for acid phosphatase (E.C. 3.1.3.2) were undertaken at pH 5.0 using alpha-naphtyl phosphate as substrate and hexazotized pararosanilin as coupling agent. After the incubations for alkaline phosphatase as well as acid phosphatase two bands showing enzyme activity were demonstrated. By means of treatment with heat (56 degrees C) prior to incubation or addition of vanadate or pyrophosphate to the incubation medium it was shown that the main part of the fast moving alkaline phosphatase band was sensitive to these procedures. The alkaline phosphatase of the slow moving band appeared to be resistant to heat or the addition of inhibitors.
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PMID:Electrophoretic separation of alkaline and acid phosphatase isoenzymes from the pulp of monkey teeth. 10 57

Cell preparations in cytochemistry are conventionally analyzed with transmitted light after fixation and reaction with agents such as azo-coupling dyes. With cell suspensions stained with fluorescent cytochemical dyes, cells can also be analyzed and sorted by flow cytometry. We have exploited the intense red fluorescence of Fast Red Violet LB generated in cytochemical reactions to perform flow cytometric analyses of alkaline phosphatase (AP) expression in rat bone marrow stromal cells. By modifying staining protocols of single-cell suspensions, we demonstrate that in comparison to staining with Fast Red TR, the method is specific, can distinguish among various levels of enzyme expression within the whole population, and permits enzyme kinetic studies of heterogeneous cell populations. The method was applied to study the effect of the glucocorticoid dexamethasone (Dx) on cell proliferation and AP expression. In low AP-expressing cells, Dx treatment at 10(-8) M increased the [3H]-thymidine labeling index from 3.85% to 5.24% (p less than 0.01). In contrast, high AP-expressing cells were unlabeled by [3H]-thymidine. The staining and analytical methods reported here facilitate the detection, isolation, and quantification of subpopulations of bone marrow stromal cells that express alkaline phosphatase activity. These experiments demonstrate the value of flow cytometry as an adjunct to conventional cytochemical methods.
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PMID:Direct flow cytometric quantification of alkaline phosphatase activity in rat bone marrow stromal cells. 137 42

We have used naphthol-ASMX-phosphate and Fast Red TR in combination with alkaline phosphatase (APase) to produce fluorescent precipitated reaction products in a non-radioactive in situ hybridization (ISH) method. To obtain optimal and discrete localization of the strongly red fluorescent ISH signals, the enzyme precipitation procedure was optimized. The optimal reaction time and the concentrations of substrate and capture agent were determined. Furthermore, polyvinyl alcohol (PVA) was used to increase the viscosity of the reaction mixture and thus to reduce diffusion of the reaction product. Our results show that the APase-Fast Red detection method has at least the same sensitivity as currently observed in other immunofluorescent detection systems. A single copy DNA sequence of 15.8 KB could be localized with high efficiency in metaphase spreads and in interphase nuclei. Double labeling procedures, in which the FITC- and azo-dye fluorescence are combined, are also feasible. The red fluorescent ISH signals showed hardly any fading as compared with FITC fluorescence on exposure to either light from the mercury-arc lamp or laser light. Therefore, these red fluorescent signals with a virtually permanent character allow a better analysis and three-dimensional localization of such cytochemically detected genomic fractions by means of confocal scanning laser microscopy as compared with the use of FITC, TRITC, or Texas Red as label.
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PMID:A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-fast red reaction. 150 67

Cyanine dye fluorescence and alkaline phosphatase activities have been compared directly by confocal microscopy in a wide variety of cells present in the follicle-associated epithelium of the mouse Peyer's patch to test the hypothesis that antigen-transporting M cells have a low membrane potential. In order to make these comparisons it was first necessary to equilibrate living tissue with the membrane potential sensitive dye DIOC5(3), fix with glutaraldehyde and then incubate the fixed tissue with naphthol AS-BI phosphate, a substrate which is hydrolysed by alkaline phosphatase present in the luminal membrane of these epithelial cells. Naphthol AS-BI produced by this reaction is then coupled to Fast Red TR diazonium salt at the site of hydrolysis. Selecting the 488 nm wavelength of the argon laser source then allows one to measure alkaline phosphatase activities as Fast Red absorbance and membrane potentials by DIOC5(3) fluorescence. Results obtained show a linear correlation between membrane potential and alkaline phosphatase activity. Relative lack of alkaline phosphatase activity, determined in fixed tissue, has been used previously to identify antigen-transporting M cells (Smith et al., 1987). The present work shows that it is now possible to recognize these cells in living tissue by measurement of DIOC5(3) fluorescence. The possible importance of this finding in providing a way to study cell surface-antigen interactions taking place in living tissue is discussed.
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PMID:Confocal microscopical analysis of epithelial cell heterogeneity in mouse Peyer's patches. 160 96

The study evaluates a novel version of the bromodeoxyuridine (BrdU) incorporation assay for in vitro proliferative responses, which works with finger-prick blood specimens. It was developed primarily for field-work involving children in Africa and elsewhere. Heparinized blood specimens from healthy volunteers were diluted 1:15 and cultured with purified protein derivative, tetanus toxoid, concanavalin A or medium alone for four-seven days and pulsed during the last 24 hours with bromodeoxyuridine. Thereafter, white and red cells were separated by Ficoll-Pacque centrifugation. The white cells were made to adhere to Multitest slides pre-treated with poly-L-lysin. After fixation with paraformaldehyde, the cells were incubated with mouse monoclonal antibodies to a surface marker for activated T cells (CD25, interleukin-2 receptor) and the reaction visualized with anti-mouse alkaline phosphatase conjugated antibodies and a Fast Red substrate. After treatment with cold acetone, the cells were covered with formamide and heated to 70 degrees C, without loss of surface staining. The incorporation of bromodeoxyuridine was visualized in the UV-microscope with mouse anti-bromodeoxyuridine monoclonal antibodies and a rabbit anti-mouse fluorescein conjugate. Both the surface staining and the nuclear fluorescence can be seen in UV-light and the cells be classified as single- or double-positive or negative. Parallel experiments on the same blood-sample from seven donors allowed for at least 42 paired comparisons between the proportion of BrdU positive cells and thymidine incorporation stimulation indices. The latter assay was carried out both in the conventional version and in a whole-blood micro-version. The rank order correlations were 0.84 and 0.91 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Whole-blood microassay for immunodetection of antigen specific cell mediated immunity using bromodeoxyuridine incorporation. 166 23

An in situ hybridization technique has been developed for the detection of immunoglobulin light chain mRNA in routine pathology specimens. The method detects kappa or lambda constant region sequences using a cocktail of synthetic oligonucleotide probes labelled with biotin or fluorescein 5-isothiocyanate (FITC) reporter molecules. The probes were labelled at flanking sites chemically by primary amine directed acylation and by 'homopolymer tailing' with terminal deoxynucleotidyl transferase using non-radioactive nucleotide analogues. The mRNA was unmasked in the formalin-fixed tissue sections by digestion with varying concentrations of proteinase K, and the hybrids were demonstrated using alkaline phosphatase with either a streptavidin/biotin based four-stage system or an anti-FITC antibody based detection system. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results confirm that the method is specific for kappa or lambda mRNA and show that specific mRNAs can be detected in routine formalin-fixed sections using non-radioactive techniques with retention of good morphology. The method reliably detects light chain mRNA in cells expressing secretory immunoglobulin. The protocol can also be applied to tissue rich in endogenous biotin by using hapten-labelled probes.
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PMID:In situ hybridization of immunoglobulin light chain mRNA in paraffin sections using biotinylated or hapten-labelled oligonucleotide probes. 212 70

An in situ hybridization technique has been developed for assessing poly(A)+ RNA preservation in routine pathology specimens. The method detects poly-adenylated RNA sequences in tissue sections using a biotinylated polydeoxythymidine (poly d(T)) probe. The probe was prepared from single-stranded 25-30 base oligo d(T) and was biotinylated using the enzyme terminal deoxynucleotide transferase with biotin-11-dUTP and dTTP in the ratio 1:4. The hybridization protocol uses varying concentrations of proteinase K to unmask mRNA sequences and the biotin-labelled hybrids are demonstrated after hybridization under standard conditions by the application of streptavidin and biotinylated alkaline phosphatase. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results have confirmed that the method is specific for poly(A)+ RNA and shows that poly(A)+ RNA can be demonstrated in routine formalin-fixed sections using non-radioactive techniques with retention of morphology. It also provides a means of optimizing the hybridization conditions for specific mRNA probes and produces a staining pattern demonstrating the relative level of poly(A)+ RNA per cell which may reveal new information about cell activity and tissue function.
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PMID:In situ hybridization demonstration of poly-adenylated RNA sequences in formalin-fixed paraffin sections using a biotinylated oligonucleotide poly d(T) probe. 247 1

A dot-immunobinding assay for the rapid and specific detection of viral antibody as well as the identification of a virus is described. Based on the use of nitrocellulose membranes for the adsorption of viral protein, this test may be used to detect the presence of virus antibody or identify an isolate within 4 to 6 hours. Use of goat anti-human IgG-alkaline phosphatase followed by a naphthol-AS-MX-phosphate: Fast Red substrate permits visual detection of a positive reaction. The use of psoralen inactivated herpes B virus permits the incorporation of this virus into the battery of test antigens.
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PMID:Dot immunobinding assay of viral antigen and antibodies. 302 39

Alkaline phosphatase immunochemical systems were evaluated for use in the demonstration of in situ hybridized biotin-labelled probes in frozen and fixed sections of tonsil. Three probes were used: total genomic DNA, pHY2.1, a human repetitive sequence which hybridizes to a 2.12 KB sequence on the Y chromosome (2000 repeats) and a 2.0 KB sequence on the autosomes (100-200 repeats), and human papilloma virus type II. Indirect, three- and five-stage detection methods were compared on cryostat sections. The indirect method involved the application of a streptavidin, biotinylated alkaline phosphatase sequence. The three-stage procedure comprised a mouse monoclonal anti-biotin, rabbit anti-(mouse immunoglobulin), mouse APAAP system. In the five-stage method the indirect and three-stage reagents were sequentially applied. Alkaline phosphatase was demonstrated using a Fast Red naphthol-capture method. The total genomic DNA probe was used initially to investigate hybridization conditions including the optimum temperature of denaturation, which was found to be higher than previously reported. The five-stage detection method gave the most sensitive results for the Y sequence probe, with intense demonstration of the Y body in male nuclei and autosomal sequences in female nuclei. This method was then applied to fixed tissue sections and gave Y body signals on Bouin's and Carnoy's fixed tissue. On the other hand tissue fixed using formalin-based solutions required proteolytic digestion as a pretreatment to hybridization for a Y body signal. The application of this methodology to viral diagnosis in routine fixed anogenital tissue and cytological preparations was also demonstrated.
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PMID:In situ hybridization: alkaline phosphatase visualization of biotinylated probes in cryostat and paraffin sections. 332 63


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