Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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 primary objective of the present study was to develop a method for quantifying the smooth muscle content of the prostate adenoma. A double immunoenzymatic staining technique was coupled with color assisted image analysis to determine the area density of the smooth muscle within the prostate adenoma. Eight males with symptomatic BPH underwent transrectal biopsy of the prostate. Four micron thick tissue sections were used for the double immunoenzymatic staining process. Rabbit anti-desmin and mouse anti-human prostatic acid phosphatase antibodies were used to selectively bind smooth muscle and prostatic epithelium, respectively. The two different tissue antigens were identified with peroxidase-antiperoxidase (PAP) and alkaline phosphatase-antialkaline phosphatase techniques. The alkaline phosphatase activity and peroxidase activity were developed with fast red and DAB chromogens. The BQ MEG IV Vista color system image analysis was used to discriminate color differences from the stained tissue sections. The thresholds were set to identify smooth muscle (dark brown), epithelium (red), fibrous tissue (pale brown), and glandular lumina (colorless). The mean area density of smooth muscle, fibrous tissue, glandular epithelium, and glandular lumina was 22%, 54%, 16%, and 9%, respectively. The present study suggests that a significant component of the prostate adenoma is smooth muscle. The application of this technique will be utilized to provide further insights into the role of smooth muscle in the pathogenesis and therapy of BPH.
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PMID:Quantifying the smooth muscle content of the prostate using double-immunoenzymatic staining and color assisted image analysis. 137 63

Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce3+ ions and the DAB-Ni-H2O2 procedure yielded better results than Ca2+ ions in the Co-(NH4)2S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce3+ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT.
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PMID:Menadiol diphosphate, a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry. 169 64

This paper describes a sequential staining procedure for double immunoenzymatic staining of pairs of antigens in frozen tissue sections and cell smears using monoclonal antibodies. This technique involves performance of an indirect immunoperoxidase sandwich (including development of the enzyme reaction) followed by an unlabelled immuno-alkaline phosphatase sandwich (the APAAP method). The two enzyme labels are revealed using DAB/H2O2 for peroxidase and naphthol AS-MX plus fast blue or fast red for alkaline phosphatase. When compared with a hapten-sandwich/biotin-avidin system, the sequential staining procedure proved to be simpler and more sensitive and was also more suitable for double immunoenzymatic staining when monoclonal antibodies were only available in small amounts. The sequential staining procedure is particularly useful for the identification of antigens distributed in different cell populations or in different sites (e.g., nucleus and cytoplasm or cell surface) of the same cell. In contrast, this method does not appear to be very suitable for demonstrating two antigens located in the same site (e.g., surface membrane) of the same cell for which purpose double immunofluorescence remains the first choice.
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PMID:Description of a sequential staining procedure for double immunoenzymatic staining of pairs of antigens using monoclonal antibodies. 243 24

A number of immunocytochemical detection systems for determining the chromosomal localization of specific nucleic acid sequences by non-radioactive in situ hybridization have been compared. The procedures were: 1. the peroxidase/diaminobenzidine (PO/DAB) combination, either or not gold/silver intensificated; 2. alkaline phosphatase marking using the nitro-blue tetrazolium plus bromochloro-indolyl phosphate substrate combination (AP/NBT + BCIP); and 3. immunogold with or without silver enhancement. The procedures were first tested and optimized in dot blot experiments and then applied to in situ hybridization. As hybridization probes, both a middle-repetitive and a unique sequence (modified with 2-acetylaminofluorene (AAF] were used. The advantages and disadvantages of the various methods for reflection contrast (RC) or transmission electron microscopic (TEM) visualization of hybrids are discussed.
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PMID:Non-radioactive in situ hybridization. A comparison of several immunocytochemical detection systems using reflection-contrast and electron microscopy. 361 Jun 73

Double immunoenzymatic labelling procedures for the localization of antigens on cells in tissue sections using horseradish peroxidase (HRP) and alkaline phosphatase have been described previously, but mainly for detecting antigens on different cells. With this type of staining when two antigens are present on the same cell, an optimal colour combination that shows a high contrast between the basic colour of each enzyme substrate product is difficult to achieve and the interpretation of their mixed colour intermediate is subjective. We present a method for the simultaneous demonstration of two antigens on the same cell. The method can be used to label either single cells in suspension, or cells in paraffin fixed tissue, using a combination of a particulate label, colloidal immunogold-silver, and an enzymatic label HRP-DAB. The method is easy to perform and utilises commercially available staining kits.
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PMID:Dual staining of lymphocyte membrane antigens with colloidal gold and biotinylated horseradish peroxidase. 750 81

The breakthrough of chemiluminescence in the field of solution immunoassays and transfer membranes prompted us to explore whether a light-based detection system could provide a gain in sensitivity over chromogenic and FITC markers for nucleic acid and protein detection on histological preparations. A Hamamatsu device and an enhanced chemiluminescence (ECL) luminol substrate of the peroxidase were used to detect epithelial and endothelial components by immunohistochemistry (IHC) and for in situ hybridization (ISH) of papilloma virus DNA. The accuracy of the signal was compared to that obtained with DAB-peroxidase, silver-enhanced DAB-peroxidase, NBT-BCIP-alkaline phosphatase, and FITC. Our results demonstrated the feasibility and high sensitivity of luminescence detection for histological preparations. In part due to the ultrasensitive videocamera and photon-counting imaging, interpretable and reproducible results were obtained within counting times shorter than 5 min, and with dilutions of the primary antibodies 100- to 10,000-fold greater than those used for chromogenic and FITC reactions. As for ISH, with identical concentrations of the HPV 18 DNA probe on HeLa cells, labeling was apparent by luminescence but undetectable with the chromogen. The morphological resolution allowed a discriminatory analysis of the signal. Therefore, at the light microscopic level, enhanced chemiluminescence offers an appealing alternative to FITC and chromogenic markers for detection and quantification of low-concentration molecules.
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PMID:Enhanced chemiluminescence: a high-sensitivity detection system for in situ hybridization and immunohistochemistry. 769 29

To visualize the enzyme Na+/K(+)-ATPase (transport ATPase) in the chick kidney and intestine two recent methods of catalytic histochemistry were modified using capture of inorganic phosphate with lead according to Mayahara et al.(1980) or cerium after Kobayashi et al. (1987). For light microscopy a new step for the visualization of the reaction product was added; lead phosphate was visualized with (NH4)2S and cerium phosphate with the DAB-H2O2-Ni-hexamonium sulfate method. Reaction product was specifically found in the basolateral plasma membrane region of enterocytes and renal epithelial cells (distal tubules, thick ascending limbs of Henle's loops, cortical collecting ducts). Treatment of the sections with 8 mM levamisole and 40 mM L-phenylalanine before and during incubation was necessary to suppress the co-reaction of non-specific alkaline phosphatase in the microvillous zone of proximal tubules and enterocytes. The reaction specificity was controlled with 10 mM ouabain which completely inhibited the basolateral activity in enterocytes and renal epithelial cells. The described methods for transport ATPase are reliable and provide reproducible results in the chick.
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PMID:Light microscopic visualization of transport ATPase in the chick kidney and intestine using catalytic histochemistry. 785 10

We describe a fast light microscopic procedure for the simultaneous enzyme cytochemical detection of three different DNA target sequences in contrasting colors in both interphase and metaphase cell preparations. Chromosome-specific DNA probes labeled with either biotin, digoxygenin, or fluorescein were hybridized as a mixture and detected clearly and accurately by precipitates of the horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color), or horseradish peroxidase-tetramethylbenzidine (PO-TMB, green color) reaction, respectively. The PO-TMB reaction product was stabilized effectively by the addition of sodium tungstate to the reaction mixture, thus making the PO-TMB reaction now generally applicable to in situ hybridization (ISH). To avoid mixing of the precipitates of the two PO reactions used in the triple-color ISH method, the first detected PO activity was always completely inactivated by a mild acid treatment before the second one was applied. Finally, the cell preparations were embedded in a thin protein layer cross-linked by formaldehyde to ensure permanent stabilization of the enzyme reaction products and optimal visualization of color contrast. The triple-color ISH detection procedure could be combined with beta-galactosidase-5-bromo-4-chloro-3-indolyl-beta- D-galactoside (beta-Gal-BCIG) immunocytochemistry (ICC), leading to the simultaneous localization of multiple DNA targets and a protein target in the same cell. The described procedure may therefore be a valuable tool in the areas of cytogenetics, cell biology, and molecular pathology.
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PMID:A novel triple-color detection procedure for brightfield microscopy, combining in situ hybridization with immunocytochemistry. 793 May 13

We describe here a simple method for combining non-radioactive and radioactive in situ hybridization and immunohistochemistry on the same brain tissue section. This approach was first developed on the well-characterized hypothalamo-neurohypophyseal system, facilitating the optimization of the triple-labeling procedure and the verification of labeling specificity. We report the simultaneous detection of vasopressin (VP) mRNA with a digoxigenin-labeled oligonucleotide, oxytocin (OT) mRNA with a 35S-labeled oligonucleotide, and OT peptide in the same 12-microns cryostat section. This was performed on floating sections as follows: first, the two probes were hybridized simultaneously; second, the peptide was detected with an immunoperoxidase-DAB procedure; third, the digoxigenin-labeled probe was detected with an alkaline phosphatase-NBT/BCIP technique; and finally, the 35S-labeled probe was detected by histological autoradiography. We also demonstrate that this approach is suitable for the simultaneous detection of tyrosine hydroxylase and two less abundant mRNAs, vasoactive intestinal peptide and vasopressin mRNAs, in the suprachiasmatic nucleus. The combination of the three techniques did not significantly diminish their specificity or sensitivity. In conclusion, this new method, permitting the simultaneous detection of three different products of gene expression in the same section, could be useful for further analysis of the phenotypic organization and its plasticity in endocrine or neural tissues.
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PMID:Combination of non-radioactive and radioactive in situ hybridization with immunohistochemistry: a new method allowing the simultaneous detection of two mRNAs and one antigen in the same brain tissue section. 809 8

The effect of voluntary exercise on 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB)-induced hepatomas was investigated in male Jc1:Wistar rats. Beginning at 10 weeks of age, animals were divided into two groups (sedentary and exercise) and housed in individual cages. Food intake and wheel exercise were automatically controlled in the cages of the exercise group. Body weights were monitored throughout the study. Food availability was controlled in order to equate length and weight gain. From 27 weeks to termination of the study at 62 weeks, all animals were administered 3'-Me-DAB in the diet at a dose level of 0.0177 g/day/kg body wt. All animals were sacrificed at 62 weeks of age. The incidence of hepatomas was significantly lower in the exercise group as compared with the sedentary group (0% and 65%, respectively). Liver weight was significantly greater in the exercise group compared with sedentary animals without hepatomas. The weight of epididymal fat pads was significantly lower in the exercise group. Serum alkaline phosphate was significantly higher in the exercise group as compared with the sedentary group. Serum gamma-glutamyl-transpeptidase levels were higher in the sedentary group than in the exercise group. In addition, gamma-glutamyl-transpeptidase levels were significantly higher in sedentary animals with hepatomas than in sedentary animals without hepatomas. These results demonstrate that voluntary exercise decreases 3'-Me-DAB-induced hepatomas and that this decrease is associated with an increase in serum alkaline phosphatase and a decrease in serum gamma-glutamyl-transpeptidase levels.
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PMID:Effect of voluntary exercise on 3'-methyl-4-dimethylaminoazobenzene-induced hepatomas in male Jc1:Wistar rats. 810 85


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