Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Digoxigenin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 613 records · Page 34Linked to original sources
PubMed1994

Kinetic microphotometric evaluation of in situ hybridization for mRNA of slow myosin heavy chain in type I and C fibres of rabbit muscle.

The present study was undertaken in order to test the possibility of microphotometric evaluation of in situ hybridizations. The histochemical detection of mRNA specific to the slow myosin heavy chain (HCI), in fibre cross sections of normal and transforming rabbit muscles with a digoxigenin-labelled complementary RNA (cRNA) probe was used as a model. Scanning densitometry of Northern blot hybridizations showed that the detection of cRNA/mRNA hybrids by a staining reaction catalysed by alkaline phosphatase coupled to an anti-digoxigenin antibody occurs in a concentration-dependent manner and follows a linear time course. These findings were the basis for elaborating a comparative microphotometric evaluation of in situ hybridization in tissue sections by measuring the reaction rate of the alkaline phosphatase-catalysed formazan production. Relative amounts of HCI mRNA were thus determined by comparing reaction rates instead of by single point microphotometry. This method was applied to studies on the distribution of HCI mRNA in different fibre types of normal rabbit muscles and and muscles undergoing fast-to-slow fibre transformation in response to low-frequency stimulation. The different fibre types were identified by histochemical staining for myofibrillar actomyosin ATPase (mATPase) in cross sections adjacent to the sections processed for in situ hybridization. On the average, type I fibres displayed 2.3-fold higher reaction rates than the mean value recorded for C fibres. According to the pronounced scattering of the values measured in single C fibres, these fibres represented a heterogeneous population in the transforming muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Sensitive detection of human growth hormone mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues by non-isotopic in situ hybridization.

A sensitive technique of non-isotopic in situ hybridization (NISH) is presented, which permits the detection of human growth hormone (hGH) mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues and human post mortem pituitaries; the latter were used as positive tissue controls in this study. In addition, a double staining procedure combining NISH and immunohistochemistry for the visualization of both hGH and hGH mRNA in the same paraffin section is described. Digoxigenin-labelled antisense hGH RNA was used for NISH of hGH mRNA. The NISH protocol was based upon an established radioactive method. Alkaline phosphatase and horseradish peroxidase-based immunoenzymatic procedures for the detection of digoxigenin-labelled RNA probes using different chromogens [4-nitro blue tetrazolium chloride (NBT), Fast Blue BB, New Fuchsin, and 3,3'-diaminobenzidine tetrahydrochloride (DAB) with or without intensification of the DAB staining] were compared. The proteolytic tissue pretreatment and the detection procedure were found to be the most critical steps for successful visualization of hGH mRNA. After optimization of the permeabilization conditions, hGH mRNA could be visualized in each case studied when alkaline phosphatase/NBT-based detection was employed. The NISH technique presented here, performed either separately or in combination with immunohistochemistry, permits retrospective analyses, of hGH (trans)gene expression in archival, paraffin-embedded specimens.

Animals↗
PubMed1994

The mapping of transgenes by fluorescence in situ hybridization on G-banded mouse chromosomes.

A highly sensitive method for the mapping of transgenes and other genes in the mouse genome is described. This technique combines high-resolution G-banding and fluorescence in situ hybridization (FISH) with either biotin/avidin-FITC or digoxigenin-anti-digoxigenin-FITC, the latter being the more sensitive. Banding patterns are obtained with trypsin/Giemsa-treated slides, and sensitivity is greatly increased by the use of mouse Cot-1 DNA. With this protocol, four different 14.5-kb human Cu/Zn-superoxide dismutase transgene insertions ranging in copy number from 2 to 8 have been localized to four different mouse chromosomes. The utility and sensitivity of this procedure were verified with a Chromosome (Chr) 16-specific cosmid probe, H22, as well as with the mapping of a high-copy-number human beta-amyloid/A4 transgene.

Animals↗
PubMed1993

Comparison of indirect and direct in-situ polymerase chain reaction in cell preparations and tissue sections. Detection of viral DNA, gene rearrangements and chromosomal translocations.

Different approaches to the in-situ polymerase chain reaction (in-situ PCR) were compared in the detection and in-situ localization of chromosomal translocations (t14; 18) immunoglobulin gene rearrangements and viral DNA (cytomegalovirus, hepatitis B-virus) in cell suspensions, cytospins and tissue sections. Single and multiple primer pairs were compared in the amplification step of indirect in-situ PCR and long genomic probes or internal oligonucleotide probes in the subsequent in-situ hybridization (ISH). For direct in-situ PCR, in which amplification products were directly labeled with digoxigenin-11-dUTP during PCR and detected immunohistochemically, only single primer pairs were used for amplification. In-situ PCR yielded best results in the cell suspensions and worked less efficiently in cytospins or tissue sections. Quantification of the results obtained in artificial cell mixtures yielded only an approximate correlation between the number of expected and observed positive cells. The specificity of the results was greater with indirect in-situ PCR than direct in-situ PCR, where false positive results were frequent. Successful indirect in-situ PCR in tissue sections required the use of multiple primer pairs for amplification and genomic probes for detection by ISH. False positive results in direct in-situ PCR were caused by primer-independent, but DNA polymerase- and cycling-dependent incorporation of digoxigenin-labeled nucleotides into cellular DNA, possibly related to DNA repair and/or internal priming. Non-specific results were most marked in tissue sections and were much less frequent in cell suspensions. In-situ PCR includes a number of different techniques, which are not equally applicable to different starting materials. Accurate interpretation of the results requires vigorous controls.

Animals↗
PubMed1976

Metabolism of digitoxin in man and its modification by spironolactone.

The effect of spironolactone on the metabolism of intravenously administered 3H-digitoxin (80 muCi) was investigated in eight patients. In three of them the labelled glycoside was given on a second occasion after spironolactone treatment had been discontinued for at least 65 days. Of total urinary radioactivity 79% was unaltered drug and 12% consisted of water soluble compounds. No digitoxigenin or digoxigenin and only trace amounts (less than 2 %) of digoxin and the bis- and monoglycosides of digoxigenin were found. After spironolactone total urinary radioactivity was unchanged but the fraction eliminated as unchanged digitoxin fell from 79 to 66 % and the water soluble compounds increased from 12 to 26 % (p less than 0.05). In addition spironolactone caused a 20 ( reduction in the half-life of serum radioactivity (p less than 0.01) and a 16 % reduction in the volume of distribution (p less than 0.05). Induction of hepatic enzymes by spironolactone is proposed to explain the alteration in the metabolism of digitoxin in man. Both the altered metabolic pattern and the reduction in the volume of distribution appear to contribute to the reduction in half-life.

Aged↗
PubMed1994

Detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization.

In situ hybridization experiments were carried out to detect avocado sunblotch viroid (ASBVd) in foliar tissue of avocado, using a digoxigenin-labelled RNA probe complementary to the ASBVd-RNA in sections of aldehyde-fixed, LRGold-embedded leaf samples. Detection of the probe was made through anti-digoxigenin antibody and protein-A colloidal gold (20 nm). Seventy to 80% of the signals came from chloroplast while the cytoplasm and vacuole were labelled with ca. 10% of the gold particles. This is in contrast with the subcellular localization of potato spindle tuber viroid and some other related viroids, which are mainly found in the nucleus.

Chloroplasts↗
PubMed1995

Zone-specific localization of cytochrome P45011B1 in human adrenal tissue by PCR-derived riboprobes.

Cytochrome P45011B1 (11 beta-hydroxylase) was detected in the human adrenal cortex and in human adenomas by in situ hybridization methods. Specific riboprobes were generated by in vitro transcription of 11 beta-hydroxylase--specific synthetic oligonucleotides with attached T7 and SP6 polymerase promotors. [35S]- and digoxigenin-labeled riboprobes were hybridized to sections of an aldosterone-producing adenoma (APA), the non-tumour portion of the corresponding adrenal gland, and two adenomas not related to hyperaldosteronism using standard protocols and varying washing conditions. After exposure of the radiolabeled sections to X-ray film, the signals were quantified and compared by statistical tests. Following autoradiography or immunohistochemical detection of the digoxigenin cytochrome P45011B1 mRNA was clearly localized in the zona fasciculata/reticularis of non-tumour portion of an human adrenal with an APA. Zona glomerulosa, medulla and connective tissue were free of label. As revealed by the semi-quantitative analysis, 11 beta-hydroxylase mRNA signals in the APA were significantly lower than those in the attached non-tumour portion and the other two adenomas. The results confirm known observations on the occurrence of cytochrome P45011B1 in the adrenal cortex of other species, but show, contrary to several immunohistochemical studies, that the enzyme is obviously not expressed in the zona glomerulosa.

Adenoma↗
PubMed1978

[A simplified radioimmunoassay for digoxin determination using a 125-j-labelled, solid-phase kit (author's transl)].

Our experience with a commercially available kit (Radioimmunoassay DIGOXIN, Boehringer, Mannheim) using (125J)-labelled digoxin and antibody-coated tubes is reported. This simplified method requires only two pepetting steps per sample and results can be obtained in 70 min. The intra- and interassay coefficient of variation ranged between 7% and 8%. The specific digoxin antibody gave no clinical relevant cross-reactions with spironolactone or prednisone (less than 0.0007%). Of the digoxin metabolites the aglucone digoxigenin showed 31% cross-reaction while the more important cardioactive metabolites digoxigenin-bis- and mono-digitoxide had the same binding affinity to the antibody as digoxin, beta-methyldigoxin and beta-acetyldigoxin. Cross-reaction with digitoxin was 6.8%. More than double-fold dilution of serum protein concentration showed little influence on the digoxin values measured. The results obtained by this new kit compare closely with those obtained by our tritium-labelled kit (r = 0.94, p less than 0.001). Therefore, the upper therapeutic limit of 1.9 ng/ml can be adopted for this method.

Antibodies↗
PubMed1996

Dot blot hybridization with a cDNA probe derived from the human calicivirus Sapporo 1982 strain.

A dot blot hybridization assay was developed for detection of human calicivirus/Sapporo/82/J (HuCV/Sa/82) or strains closely related to HuCV/Sa/82 in stool specimens. The cDNA derived from the RNA-dependent RNA polymerase (RDRP) region of HuCV/Sa/82 was used as a positive probe and the pBR322 DNA as a negative control probe. Both probes were labeled with digoxigenin and the products of hybridization reaction were detected with an anti-digoxigenin antibody-alkaline phosphatase conjugate. This assay was specific for HuCV/Sa/82 and for HuCV antigenically related to HuCV/Sa/82. The lower limit of sensitivity of this assay was estimated to be about 10(5) physical particles or 10 pg of cDNA, similar to that of the previously developed ELISA for HuCV. In 1273 stool specimens obtained from children with acute gastroenteritis in Sapporo, Japan, 110 (8.6%) contained small round structured viruses by EM and 23 (1.8%) were positive for HuCV antigenically related to HuCV/Sa/82 by either the hybridization assay or ELISA. A higher positive rate was obtained with the dot blot assay (21%) than by ELISA (10%), suggesting that the dot blot assay either detects HuCV more broadly than the ELISA or detects HuCV covered with fecal antibodies which interrupt antigen-antibody reactions in the ELISA. Negative results for detection of Norwalk virus (NV) cDNA and feline calicivirus (FCV) RNA by both this assay and the ELISA indicated that the HuCV/Sa/82 strain is distinct antigenically and genetically from NV and FCV.

Acute Disease↗
PubMed1989

Detection of tetracycline-resistant strains of Ureaplasma urealyticum by hybridization assays.

Five wild-type strains of tetracycline-resistant Ureaplasma Ureaplasma urealyticum and one tetracycline-sensitive Ureaplasma urealyticum strain were tested for presence of the tetM sequence by hybridization assays. Two sorts of probes were used, a plasmid containing the tetM fragment and the tetM fragment alone. Two sorts of label were used for each, digoxigenin and radio-labelling. Both purified DNA at various concentrations and non-purified DNA in the pellet from a 10 ml culture were used as targets. The digoxigenin-labelled whole plasmid probe was as specific as the insert alone when hybridized with non-purified DNA, and was more sensitive than radio-labelled probes.

DNA Probes↗
PubMed1994

Detection of amplified Chlamydia trachomatis DNA using a microtiter plate-based enzyme immunoassay.

A microtiter plate-based method to detect amplified DNA was developed. The method uses on biotin-labeled primer in the polymerase chain reaction (PCR) mixture. The labeled amplicon is bound to streptavidin-coated microtiter plates, denatured and hybridized to a digoxigenin-labeled probe. The specificity of the hybridization reaction was optimized by varying the temperature of the subsequent washing step and adding urea to the washing buffer. The digoxigenin label was detected using an enzyme immunoassay (EIA). This PCR-EIA was compared with a standard PCR assay that uses gel electrophoresis, blotting and hybridization to detect the amplicon, with isolation in cell culture, and with an antigen detection EIA (Chlamydiazyme) in the diagnosis of Chlamydia trachomatis infection in 309 female patients attending a sexually transmitted diseases outpatient clinic. The prevalence of Chlamydia trachomatis infection as determined by isolation in cell culture, EIA, PCR-EIA and standard PCR assay was 9.1%, 8.7%, 12.3%, and 12.9%, respectively. Compared with results of a reference set of confirmed-positive cases (defined by a positive result in two or more independent assay after analysis of discrepancies), the sensitivity and specificity was 71.1% and 99.6% for cell culture, 65.8% and 99.3% for the EIA, 92.1% and 98.9% for the PCR-EIA, and 97.4% and 98.9% for the standard PCR assay. It is concluded that the PCR-EIA described is a fast, sensitive and specific method for detecting Chlamydia trachomatis in clinical specimens.

Amino Acid Sequence↗
PubMed1997

Expression of connexin43 mRNA in adult rodent brain.

The expression of connexin43 mRNA was detected in adult rat brains by in situ hybridization methods. Specific digoxigenin riboprobes were generated by in vitro transcription of two PCR-amplified fragments of connexin43 cDNA which lack homology with any other published connexin. Following immunohistochemical detection, the digoxigenin cRNA was found to occur in various neuronal populations including Purkinje cells of the cerebellum, pyramidal cells of the neocortex and the hippocampal formation, as well as granule cells of the dentate gyrus and various neurons of diverse hindbrain nuclei. This ubiquituous expression of connexin43 mRNA in adult neurons, in particular in neocortical pyramidal cells, is surprising insofar as gap junction communication in adult bains has been considered to be confined to specific subpopulations of neurons revealing a high incidence of synchronized electrical activities in contrast to the postnatal brain where interneuronal coupling via gap junctions precedes the formation of chemical transmission. In addition, connexin43 is regarded as being preferentially expressed in astrocytes, although its presence in adult neurons has not definitely been excluded. We propose that adult neurons preserve their capability of expressing functional gap junctions more frequently than presently considered and that connexin43 is a most likely neuronal gap junction protein candidate.

Animals↗
PubMedDate not supplied

Simultaneous triple fluorescence detection of mRNA localization, nuclear DNA, and apoptosis in cultured cells using confocal scanning laser microscopy.

We describe a multifluorescence labeling technique for simultaneous detection of mRNA, nuclear DNA, and apoptosis in cultured cells. Digoxigenin-labeled cRNA probes were used to study proto-oncogene expression in rat pleural mesothelial cells undergoing apoptosis following exposure to crocidolite asbestos or hydrogen peroxide (H2O2). Hybridized cRNA probe was detected by immunolocalization with an anti-digoxigenin monoclonal primary and fluorophore-conjugated anti-mouse secondary antibody. Cells undergoing apoptosis were simultaneously identified by the TdT-mediated biotin-dUTP nick-end labeling (TUNEL) method and a streptavidin-conjugated far-red fluorophore, and nuclear DNA was stained with oxazole yellow dimer (YOYO-1). With confocal scanning laser microscopy, we demonstrated increased c-jun mRNA expression within the cytoplasm of both TUNEL-positive and non-apoptotic cells following exposure to either crocidolite asbestos or H2O2. Thus, this technique represents a useful in vivo approach for evaluating apoptosis-associated gene expression with confocal scanning laser microscopy.

Animals↗
PubMed1998

Mapping of D1 dopamine receptor mRNA by non-radioactive in situ hybridization.

In order to improve the identification and characterization of dopaminoceptive neurons, the rat brain was mapped for D1 dopamine receptor mRNA by non-radioactive in situ hybridization (ISH) with a 45mer digoxigenin-labeled oligonucleotide probe. The specificity of the results was controlled with the help of a 396-bp D1 receptor riboprobe. Labeled hybrids were visualized with an alkaline phosphatase-coupled anti-digoxigenin antibody. The high resolution obtained permitted individual labeled cells to be identified and to distinction between cell bodies and processes. D1 mRNA was largely confined to neurons. With the exception of ependymal cells, glial cells were not distinctly labeled. Subcellularly, D1 mRNA was localized to perikarya but not to dendrites or axons. D1 receptor-expressing neurons were present in all of the known terminal fields of mesencephalic or diencephalic dopaminergic neurons. However, D1 message was also detected in brain areas which are not known to contain D1 ligand binding sites or in which the presence or the cellular source of this receptor subtype had previously not been unequivocally established, such as the hippocampus or cerebellar cortex. Moreover, labeled neurons were present in regions not known to receive dopaminergic projections, such as the thalamic and some brainstem nuclei. We conclude that this ISH technique provides a considerable gain in sensitivity and resolution with regard to neurotransmitter receptor mapping.

Animals↗
PubMed1999

Localization in situ of type VI collagen protein and its mRNA in mesangial proliferative glomerulonephritis using renal biopsy sections.

Extracellular matrix accumulation is crucial in the pathogenesis of glomerulosclerosis in mesangial proliferative glomerulonephritis (GN). In an attempt to explore the distribution of type VI collagen and its synthesizing cells in normal and diseased glomeruli, we investigated mRNA and protein expression of type VI collagen in renal biopsy sections, histologically diagnosed as mesangial proliferative GN. Five renal biopsies from patients diagnosed as having minor glomerular abnormalities and one surgical renal tissue were also simultaneously examined as controls. Immunohistochemical studies revealed type VI collagen immunostaining in the mesangium and glomerular basement membrane of the control glomeruli. Compared to the control, increased deposition of type VI collagen was noted in the mesangial proliferative and sclerotic lesions in GN. To identify the cells responsible for the synthesis of type VI collagen mRNA, renal sections were hybridized in situ with digoxigenin-labeled antisense oligo-DNA probe complementary to a part of alpha1 (VI) mRNA. Occasionally intraglomerular cells hybridized with digoxigenin-labeled antisense pro alpha1 (VI) oligo-DNA in control glomeruli. An increased number of intraglomerular cells (mostly epithelial cells) were, however, positive for alpha1 (VI) mRNA expression in GN sections. The present study documents the distribution of type VI collagen in the normal glomeruli and provides further evidence of accelerated synthesis of this collagen in mesangial proliferative GN.

Adolescent↗
PubMed1998

Rat fetuin: distribution of protein and mRNA in embryonic and neonatal rat tissues.

Fetuin is a serum protein widely distributed in the animal kingdom and found in all mammalian species so far investigated. It is mainly a fetal protein, in the sense that the highest concentrations are found in serum and body fluids of embryos and fetuses. In order to elucidate possible biological functions of fetuin, we have studied its synthesis and distribution during the prenatal development of the rat with immunohistochemistry and in situ hybridization. We have isolated fetuin from rat serum and produced an antibody against this protein. In situ hybridization was performed using a 375-nucleotides-long digoxigenin-labeled riboprobe. Fetuin was unevenly distributed in all organ systems during development, with the most pronounced expression at E 10Fetuin is a serum protein widely distributed in the animal kingdom and found in all mammalian species so far investigated. It is mainly a fetal protein, in the sense that the highest concentrations are found in serum and body fluids of embryos and fetuses. In order to elucidate possible biological functions of fetuin, we have studied its synthesis and distribution during the prenatal development of the rat with immunohistochemistry and in situ hybridization. We have isolated fetuin from rat serum and produced an antibody against this protein. In situ hybridization was performed using a 375-nucleotides-long digoxigenin-labeled riboprobe. Fetuin was unevenly distributed in all organ systems during development, with the most pronounced expression at E16-E18. Fetuin expression was present in germinal cell populations, e.g., in the basal layer in the skin, in the germinal cell populations in the brain anlage and the gonads, and it was heavily expressed in the fetal hemopoietic liver. Furthermore, fetuin was expressed in the gastrointestinal epithelium prior to the development of glands and crypts. Fetuin was widely distributed in mesenchymal derived tissues, e.g., bone and muscle. In the developing kidney fetuin was heavily expressed is both mesenchymal condensations and glomerular anlages. Thus, fetuin was located in cells or structures undergoing differentiation and transformation. As fetuin has been shown previously to interfere with hormone signaling of transforming growth factor-beta, insulin and hepatocyte-growth factor, fetuin might be involved in cell differentiation and tissue transformation during the initial histogenesis, i.e., the time period in which cellular phenotypic characteristics are established.

Animals↗
PubMed1998

Immunohistochemistry and in situ hybridization of the androgen receptor in the developing human prostate.

UNLABELLED: As it is suggested that the androgen receptor mechanism is required for prostatic development, we attempted to determine the appearance, expression and distribution of the androgen receptor in embryonic, infantile and pubertal human prostate. Using mono- and polyclonal antibodies and a digoxigenin-labeled 713 bp riboprobe, the androgen receptor expression in paraffin sections of fetal, infantile, and pubertal prostates was studied at the protein and RNA level. Under highly standardized conditions, application of the polyclonal antibodies resulted in a weak cytoplasmic and nuclear labeling of the epithelium of fetal glands. No immunoreaction was obtained with monoclonal antibodies. Applying the polyclonal antibody to pubertal and adult specimens, immunoreactivity of the androgen receptor was positive in nuclei of adluminal and basal epithelial cells, in interstitial and vascular smooth muscle cells and vascular endothelium, whereas ganglionic cells and enteroendocrine cells were negative. In situ hybridization with the digoxigenin-labeled riboprobe gave clear positive results already in epithelium of very young fetal specimens. A semiquantitative visual evaluation of in situ hybridizations showed that intermediate intensity of expression was increased in pubertal and adult specimens, whereas strong expression was reduced in prostatic epithelium. CONCLUSIONS: The essential findings are: (1) an early expression of androgen receptor mRNA in the fetal prostate; (2) no immunoreaction of monoclonal antibodies against the androgen receptor in the same specimens, (3) a decrease of androgen receptor mRNA expression, but increase in immunoreactivity of the androgen receptor protein with the onset of glandular maturation during puberty.

Adolescent↗
PubMed1996

The autoantigen La/SS-B: analysis of the expression of alternatively spliced La mRNA isoforms.

The gene for the nuclear autoantigen La/SS-B encodes two La mRNA isoforms. In order to study the function and expression of both La mRNA forms, an in situ hybridization procedure was developed allowing the selective identification of either exon 1 or exon 1'. For this purpose, digoxigenin-labeled exon-specific sense and anti-sense probes were prepared by in vitro transcription from plasmids that contained the respective exon sequence. Detection of the probes was carried out by using rhodamine-conjugated anti-digoxigenin antibody and confocal laser scanning microscopy. Both La mRNAs were found in the cytoplasm of endothelial cells but not in smooth muscle cells. In addition to the in situ technique, an assay system was established allowing the expression ratio of the two mRNA forms to be determined. The estimation was based on the amplification of exon 1 and 1' La cDNAs in parallel by using a three primer polymerase chain reaction. The ratio of the exon 1 to exon 1' La mRNA forms was determined to be about 5:1 in liver tissue and endothelial cells. The data support the conclusion that both La mRNA forms represent finally processed cytoplasmic mRNAs that are up- or downregulated in parallel.

Adult↗