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)

A murine monoclonal antibody specific for calf intestinal alkaline phosphatase has been prepared and used in an unlabeled antibody bridge technique for labeling monoclonal antibodies. This procedure--the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) method--gives excellent immunocytochemical labeling of tissue sections and cell smears, comparable in clarity and intensity to that achieved with immunoperoxidase labeling. If the enzyme label is developed with a naphthol salt as a coupling agent and Fast Red or hexazotized new fuchsin as a capture agent, a vivid red reaction product is obtained which is very easily detected by the human eye. For this reason the APAAP technique was found particularly suitable for labeling cell smears (for both cytoplasmic and surface-membrane antigens) and for detecting low numbers of antigen-bearing cells in a specimen (e.g., carcinoma cells in a malignant effusion). It was found possible to enhance the intensity of the APAAP labeling reaction substantially by repeating the second and third incubation steps (i.e., the unlabelled "bridge" antibody and APAAP complexes). The APAAP technique was superior to immunoperoxidase labeling for staining tissues rich in endogenous peroxidase, and could be used in conjunction with immunoperoxidase methods for double immunoenzymatic staining. The method was also applicable to the detection of antigenic molecules following their electrophoretic transfer from SDS-polyacrylamide gels to nitrocellulose sheets ("immunoblotting").
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PMID:Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). 619 55

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 the development and application of a sensitive high-resolution fluorescence alkaline phosphatase (APase)-Fast Red immunocytochemical (ICC) staining method in combination with fluorescence in situ hybridization (ISH) and bromodeoxyuridine (BrdU) detection. The high fluorescence intensity, accurate localization, and advantageous slow-fading properties make the APase-Fast Red reaction a valuable tool for detection of antigens or specific DNA probes in biological cell preparations. Since the enzyme precipitate proved to be resistant to enzymatic pre-treatment steps and stable during the entire ISH procedure, APase-Fast Red immunostaining could be combined with subsequent visualization of DNA target sequences by fluorescence ISH. The lung cancer cell lines NCI-H82 and EPLC 65 were used as a model system for simultaneous detection of cell proteins, such as the neural cell adhesion molecule (N-CAM), cytokeratin filaments, lamin or the Ki67 antigen (Ki67-Ag), and centromere-specific DNA probes for human chromosomes 1, 7, or 17. In addition, the combined ICC/ISH procedure could be extended with the immunodetection of BrdU incorporated by tumor cells in S-phase. As a consequence, a combined ICC/ISH/BrdU detection procedure is now available that enables analysis of relatively complex tumor populations on the basis of different ICC and genetic markers as well as proliferative activity.
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PMID:Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells. 801 80

Quantitative techniques in immunohistochemistry are needed, but they are rarely applied because of doubtful reproducibility. We have developed a method for the detection of collagen types I and III in situ. The method applied was a two-step immuno-alkaline phosphatase technique with visualization of the end-product with Fast Red. The staining intensity was measured with a microdensitometer and the results expressed as ratios. The method yielded results that were unaffected by variations in tissue section thickness but which were proportionally related to time and antigen concentrations. Leiomyoma tissue, with a ratio of collagen types I and III of approximately 1.0, was used to establish the appropriate dilutions of the antibodies, thus assuring identical optical densities. By having the leiomyoma tissue sections incubated together with the heart tissue specimens, leiomyoma tissue was also helpful in correcting deviations from the 1.0 ratio. Accurate measurements of collagen type I/III ratios in normal human heart specimens were obtained with the present quantitative immunohistochemical technique.
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PMID:Quantification in immunohistochemistry: the measurement of the ratios of collagen types I and II. 804 7

Procedures that combine immunocytochemistry (ICC) and in situ hybridization (ISH) techniques are now used to investigate phenotype/genotype relationships in the same cells. In this report we describe three rapid procedures for simultaneous detection of a nuclear antigen, progesterone receptors (PR), and the centromeric region of chromosome 11 (to which the human PR gene has been assigned) in T47-D cells. Proteins were stained by precipitates of horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color) or alkaline phosphatase-nitroblue tetrazolium-X-phosphate (APase-NBT-X-Phosphate, blue color) respectively. To obtain a suitable contrast for the two labels, we detected DNA on PO-DAB and APase-NBT-X-phosphate-immunostained cells with interphasic fluorescent in situ hybridization (FISH). By contrast, we combined the APase-Fast Red ICC with an immunocytochemical ISH using alkaline phosphatase-NBT-X-phosphate detection. Only the procedure combining APase-NBT-X-phosphate ICC and FISH ensures optimal visualization of both the PR content and the number of chromosome 11. This method easily provides simultaneous localization of DNA and protein targets in the same cells and should be applicable to many other situations.
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PMID:Methods for simultaneous interphase in situ hybridization and nuclear antigen immunocytochemistry in T47-D cells. 860 77

The 7TD1 B-cell hybridoma was found to spontaneously express alkaline phosphatase (ALP), an enzyme which is produced by splenic B lymphocytes once optimally activated. Determination of ALP levels during cell growth and departure to apoptosis showed fluctuations. Following a temporary increase within the first 24 h, enzyme expression was maintained at high levels during the early proliferation stage, and then declined from 3 to 4 days in mid-exponential phase to basal levels at day 6 when living cells were no longer detectable and the apoptotic process was completed. The protein synthesis inhibitor, cycloheximide (1 microg/ml), decreased ALP production while stimulating a strong apoptosis of 7TD1 cells, within 4 h. Aphidicolin (1 microg/ml) maintained ALP production and provoked a release of ALP activity into the surrounding medium; it also induced apoptosis, but with a 24 h delay. Quantification of apoptosis and ALP expression by flow cytometry, after simultaneous staining of DNA with Hoechst 33342 and ALP with naphthol AS-TR phosphate/Fast Red RC fluorescent reagent, revealed cell cycle modulation of ALP expression, its activity increasing as 7TD1 cells progressed from G1 phase into S and G2/M phases of the cell cycle in control as well as in drug-treated cells. Kinetics of drug-induced apoptosis and higher expression of ALP associated preferentially with active cell growth during the prevention stage of apoptosis suggested a possible link between cellular ALP expression and cell survival.
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PMID:Expression of alkaline phosphatase by a B-cell hybridoma and its modulation during cell growth and apoptosis. 874 13

We describe a new application of a bright-field microscopic procedure for rapid enzyme cytochemical detection of repeated DNA sequences in metaphase preparations and frozen tissue sections. Various chromosome-specific oligonucleotide primers were used in up to three sequential primed in situ (PRINS) labeling reactions together with Taq DNA polymerase and biotin, digoxigenin and/or fluorescein isothiocyanate (FITC)-modified nucleotides. DNA target sequences were localized simultaneously by the precipitates of the horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color) and horseradish peroxidase-teramethylbenzidine (PO-TMB, green color) reaction in hematoxylin counterstained metaphases and interphase nuclei using a standard bright-field microscope. In addition, a protocol is reported for the application of PRINS to frozen tissue sections from normal colon and bladder epithelium. Methanol/acetic acid fixation in combination with a pepsin digestion before performing the PRINS reaction proved to be critical steps in the total procedure that permits access of the PRINS reactants, while preserving the morphology of the nuclei in the tissue. Quantification of PRINS signals showed the majority of epithelial cells with the expected two chromosome copies. The described procedures can be considered valuable tools for application in molecular cytogenetics, cell biology and pathology.
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PMID:Rapid bright-field detection of oligonucleotide primed in situ (PRINS)-labeled DNA in chromosome preparations and frozen tissue sections. 882 52

Visualisation and localisation of specific DNA sequences were performed by fluorescence in situ hybridisation (FISH), confocal laser scanning microscopy (CLSM), and factor analysis of biomedical image sequences (FAMIS). HeLa cells containing 10-50 copies per cell of human papillomavirus (HPV) DNA type 18 integrated in cellular DNA were used as a model. HPV-DNA was identified by DNA probes and DNA-DNA hybrids were revealed by alkaline phosphatase and Fast Red (FR) TR salt/naphtol-MX phosphate. Cell nuclei were counterstained with thiazole orange (TO). FAMIS summarises image sequences into a reduced number of images called factor images and curves called factors. Factor images correspond to spatial distributions of the different factors. Factors estimate different individual physical behaviours in the sequence (extinction velocity, spectral emission, depth emission profiles). We verified that HPV-DNA hybridisation signals are specific to the spectrum of FR, and distinguished between FR and TO. The latter result was found by taking into account differences in their extinction velocities. The focus of CLSM was improved on 3D image sequences, and the location of fluorescent signals inside the preparations was determined. Factor images showed that FR stained targets were located on different focal planes at the periphery of the nuclei.
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PMID:Laser scanning confocal microscopy and factor analysis of biomedical image sequences (FAMIS) to detect and characterise HPV DNA sequences by FISH in HeLa cells. 926 46

Human papillomavirus (HPV) infection with potentially oncogenic types 16 or 18 is common in genital lesions especially in uterine carcinomas. In such lesions, in situ hybridization with non-radioactive probes is a powerful tool for the histopathologist to detect and type HPV DNA either on cell deposits or on tissue sections. The use of an immunohistochemical method involving alkaline phosphatase and Fast Red TR salt/naphthol AS-MX phosphate is proposed for use with conventional bright-field or fluorescence microscopy as well as by laser scanning confocal microscopy. The alkaline phosphatase-Fast Red reaction has the advantage of producing a red precipitate that permits the detection of in situ hybridization signals by bright-field microscopy, and of obtaining a strong red fluorescence characterized by a lack of bleaching when excited by a green light. Therefore, the alkaline phosphatase-Fast Red reaction is well adapted for observations by fluorescence and confocal microscopy, the latter method allowing the detection, in tissue sections of cervical intraepithelial lesions, of small punctate and large diffuse hybridization signals, considered as integrated and episomal states of HPV DNA respectively. The combination of in situ hybridization with the alkaline phosphatase-Fast Red reaction and confocal microscopy is particularly convincing when hybridization signals are of small size and/or of low fluorescence intensity, especially if they are present in various focal planes; in such conditions, infected cells are easily detected by three-dimensional reconstruction. Therefore, this combination is a suitable method for identifying and characterizing HPV DNA in cells and tissue sections.
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PMID:Detection of human papillomavirus DNA in genital lesions by enzymatic in situ hybridization with Fast Red and laser scanning confocal microscopy. 927 57

Visualization and localization of specific DNA sequences were performed by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM), and four-dimensional factor analysis of biomedical image sequences (4D-FAMIS). HeLa and SiHa cells containing, respectively 20-50 and 1-2 copies per cell of human papillomavirus (HPV) DNA type 18 and 16 integrated in cellular DNA were used as models. HPV-DNA was identified using DNA probes containing the whole genome of HPV-DNA type 18 or 16, and DNA-DNA hybrids were revealed by alkaline phosphatase and Fast Red. Cell nuclei were counterstained with thiazole orange (TO) or TOTO-iodide. 4D image sequences were obtained using successive dynamic or spectral sequences of images on different optical sections from CLSM. The location of fluorescent signals within the preparations was determined by FAMIS. This original method summarizes image sequences into a reduced number of images called factor images, and curves called factors. Factors estimate different individual physical behaviours in the sequence such as extinction velocity, spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. We distinguished between Fast Red and nucleus stainings in HPV-DNA hybridization signals by taking into account differences in their extinction velocities (fluorescence decay rate) or spectral patterns, and in their focus (depth emission profiles). In HeLa cells, factor images showed that Fast-Red-stained targets could be distinguished from nucleus stainings, and were located on different focal planes of the nuclei. In SiHa cells, 4D-FAMIS determined as few as 1-2 copies per cell of HPV-DNA type 16 located in continuous focal planes. Therefore, 4D-FAMIS, together with CLSM, made the detection and characterization of low copy numbers of genes in whole cells possible.
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PMID:Four-dimensional factor analysis of confocal image sequences (4D-FAMIS) to detect and characterize low copy numbers of human papillomavirus DNA by FISH in HeLa and SiHa cells. 1019 2


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