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PubMed1992

Chick heart cells with high intracellular calcium concentration have a higher affinity for cardiac glycosides than those with low intracellular calcium concentration, as revealed by affinity labelling with a digoxigenin derivative.

Digital-imaging fluorescence microscopy with fura-2 allows the determination of intracellular calcium concentration ([Ca2+]i) in single cells. At a cell density of 10(5) cells/petri dish 44% of the chick embryo heart cells had a high [Ca2+]i of 99.4 +/- 7.1 nM and 56% of the cells a low [Ca2+]i of 27.8 +/- 4.4 nM (mean +/- SE). This laboratory previously reported that high-[Ca2+]i and low-[Ca2+]i cells from chick embryo hearts differ in their sensitivity to cardiac glycosides, as shown by measuring the increase in [Ca2+]i to reach a new steady state [Ahlemeyer, B., Weintraut, H., Seibold, G. & Schoner, W. (1991) in The sodium pump: recent developments (Kaplan, J. H. & De Weer, P., eds) pp. 653-656, Rockefeller University Press, New York]. This time we used N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) which binds irreversibly to amino groups of the Na+/K(+)-ATPase, and sheep anti-digoxigenin Fab fragments coupled with fluorescein isothiocyanate to identify different cardiac glycoside-binding sites. Half-maximal labelling of high-[Ca2+]i cells was obtained at 0.36 nM HDMA, and at 12.0 nM with the low-[Ca2+]i cells. Specific labelling of the cells by HDMA was 91% and 80% in high-[Ca2+]i and low-[Ca2+]i cells, respectively, as revealed by competition experiments with a 1000-fold excess of ouabain. HDMA half-maximally elevated the [Ca2+]i of high-[Ca2+]i cells at a concentration of 50 pM and that of low-[Ca2+]i cells at 8.0 nM. Concentrations higher than 0.1 microM produced signs of intoxication. When the labelled cells were subjected to a SDS/PAGE, a 100-kDa band was found to contain HDMA. The electrophoretic mobility of a protein labelled at 10 nM HDMA was slightly higher than that of a protein labelled at 1.0 microM. The data suggest that different isoforms of the alpha-subunit of Na+/K(+)-ATPase may exist in low-[Ca2+]i and high-[Ca2+]i cells of chick embryo heart.

Affinity Labels↗

PubMed1992

Detection of cytomegalovirus by in situ hybridization using a digoxigenin-tailed oligonucleotide.

A non-isotopic in situ hybridization procedure was used to detect cytomegalovirus (CMV) sequences within routinely fixed tissue. A digoxigenin-tailed oligonucleotide was hybridized to sections of specimens obtained at autopsy from 2 patients with CMV infection. Hybrids were revealed by an alkaline phosphatase-conjugated anti-digoxigenin antibody. Serial sections were also assayed for the presence of CMV by in situ hybridization with a biotin-labelled cDNA probe and by immunohistochemistry and routinely stained for morphological evaluation. Results show that the two in situ hybridization procedures are equally sensitive but superior to the immunohistochemical detection of the viral antigen. Most cells positive for CMV DNA had the cytopathological features characteristic of CMV infection. A minor population of infected cells lacking morphological changes was also found. We recommend the routine application of the oligonucleotide-based assay because it is specific, easy and less expensive than other similar procedures.

Alkaline Phosphatase↗

PubMed1991

Rapid confirmation of Listeria monocytogenes isolated from foods by a colony blot assay using a digoxigenin-labeled synthetic oligonucleotide probe.

An oligodeoxyribonucleotide probe based on the sequence of a 321-bp internal fragment of the msp gene encoding a major secreted polypeptide of Listeria monocytogenes was labeled with digoxigenin by using terminal deoxynucleotidyl transferase. The specificity of the digoxigenin-labeled probe was determined by dot blot assays. The probe reacted with all strains of L. monocytogenes tested (12 of 12 strains representing five serotypes). The probe did not react with any other Listeria species or with other gram-positive bacteria (Brochothrix, Erysipelothrix, Corynebacterium, Rhodococcus, Lactobacillus, Leuconostoc, Bacillus, Staphylococcus, and Streptococcus). The probe was used to develop a colony blot assay for the rapid confirmation of L. monocytogenes on Listeria-selective agars which had been streaked with food enrichment cultures. Forty-eight food samples were tested by conventional culture and DNA colony blot assay. The sensitivity and specificity of the DNA colony blot were 100 and 97%, respectively.

Bacterial Proteins↗

PubMed1990

Identification of enterotoxigenic Escherichia coli by colony hybridization with nonradioactive digoxigenin-labeled DNA probes.

Enterotoxigenic Escherichia coli (ETEC) strains were readily identified in pure and mixed cultures with nonradioactive, digoxigenin-labeled DNA probes coding for heat-labile (LTI) and heat-stable (STaI, STaII, and STb) enterotoxins. Digoxigenin-labeled ETEC fragments were more sensitive than and exhibited less nonspecific background contamination than biotinylated ETEC probes.

Animals↗

PubMed1992

Detection of pathogenic Yersinia enterocolitica by polymerase chain reaction and digoxigenin-labeled polynucleotide probes.

Yersinia enterocolitica is widespread in nature, but only a few bioserotypes are involved in human infections. Pigs are considered to be the major reservoirs of pathogenic strains. It is essential to have an accurate and rapid method for the detection of pathogenic yersiniae. To achieve this objective, 19-base synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) to detect the ail gene (which is conserved only in pathogenic strains) in strains of Y. enterocolitica and related species originating from pigs or pork products. Digoxigenin-labeled probes derived from the ail, inv, and yst genes were also evaluated on these strains. The PCR amplified a 273-bp fragment of the ail gene involved in eukaryotic cell invasion and serum resistance. The PCR detected template DNA only in strains of Y. enterocolitica traditionally classified as human pathogens but not in biotype 1A strains and related species. Other members of the family Enterobacteriaceae were also negative for the target gene. The digoxigenin-labeled ail probe gave identical results to the PCR. By use of this nonisotopic method, inv-homologous DNA was detected only among yersiniae, except for Y. ruckeri. Although all pathogenic serotypes of Y. enterocolitica were positive for the heat-stable enterotoxin yst gene, two strains of biotype 1A, one Y. intermedia strain, and six other species of the Enterobacteriaceae were also positive. Our results support the notion that pigs constitute an important reservoir of pathogenic Y. enterocolitica and that the inv-homologous sequence is Yersinia specific.

Bacteria↗

PubMed1991

Interphase cytogenetics using biotin and digoxigenin labelled probes: III. Increased sensitivity and flexibility for detecting HPV in cervical biopsy specimens and cell lines.

A monoclonal antibody to digoxin enabled sandwich techniques to be used for the detection of hybridised digoxigenin labelled probes in cultured cells and paraffin wax sections. This system has greater flexibility than alkaline phosphatase conjugated polyclonal antidigoxigenin antibody and permits the use of alternative detector enzymes, such as horseradish peroxidase and fluorescence labels. The APAAP detection system that does not require the use of biotin can also be used in situations where endogenous biotin is a problem. The low level of background staining combined with precise substrate deposition of the amplified peroxidase system gives higher sensitivity and resolution. This permits localisation of closely adjacent chromosomal loci in interphase nuclei. The most sensitive peroxidase based digoxigenin detection system visualises two and a half to 12 copies of human papillomavirus (HPV) per nucleus. This system is also suitable for the analysis of low copy number HPV infection of cervical tissues.

Alkaline Phosphatase↗

PubMedDate not supplied

Comparison of four in situ hybridization methods, based on digoxigenin- and biotin-labelled probes, in detecting HPV DNA in male condylomata acuminata.

We have compared the efficacy of digoxigenin- and biotin-labelled probes in detecting HPV DNA by in situ hybridization on paraffin-embedded tissue sections of 57 male condyloma-suspect genital lesions. Each biopsy was hybridized with at least three of the following four methods: digoxigenin-labelled HPV DNA probes (Dig-HPV), biotinylated HPV-DNA probes (Bio-HPV), and two commercial methods (ViraType in situ and PathoGene), both based on biotinylated DNA probes. The hybridization products were visualized with colourigenic enzyme substrates. In most biopsies, the 4 methods gave equal results although cross-hybridization was most often found with the low-stringency ViraType method. Dig-HPV 6/11 probes gave positive results about twice as often as either of the commercial methods. No such difference, however, was found for HPV 16/18 probes. DNA of any type of HPV 6/11, 16/18 or 31/33/35 or 51 was detected in 28/43 (65%) of lesions showing condyloma acuminatum histology but in none of the 14 biopsies with no histological signs of HPV infection. In HPV-positive condylomata with no cellular atypia. HPV 6/11 was detected in 87% (13/15), and HPV 16/18 in 27% (4/15). In biopsies with cellular atypia, HPV types 6/11 were detected in 62% (8/13), HPV types 16/18 in 46% (6/13), and HPV types 31/33/35 or 51 in 50% (6/12). In about 50% of the biopsies where at least one hybridization method gave a positive result, either one of the commercial methods gave a negative result.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1996

Detection of porcine reproductive and respiratory syndrome virus in cell cultures and formalin-fixed tissues by in situ hybridization using a digoxigenin-labeled probe.

A nonradioactive in situ hybridization method is described for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in cell cultures and in formalin-fixed paraffin-embedded tissue sections originating from experimentally infected pigs and from 1 field case. A 174 bp cDNA probe targeting the viral RNA encoding the nucleocapsid protein of a Canadian PRRSV isolate was generated by polymerase chain reaction. The cDNA probe was labeled by random priming with digoxigenin-dUTP using a commercially available kit. The ability of the digoxigenin-labeled probe to specifically detect PRRSV RNA was tested on cultured cells infected with 6 Canadian PRRSV isolates, a US PRRSV isolate and the European Lelystad isolate. The probe detected all Canadian PRRSV isolates tested as well as the US PRRSV isolate but did not detect the Lelystad isolate. In addition, when tested on formalin-fixed paraffin-embedded tissue sections from pigs experimentally infected with several Canadian isolates and from a field case, a strong signal without background staining was obtained. Our results indicate that nonradioactive in situ hybridization could represent a useful tool for the detection of PRRSV in routinely fixed and processed tissues. In situ hybridization could also be used to differentiate infection by North American and European Lelystad-like PRRSV isolates.

Animals↗

PubMed1993

A method for simultaneous detection of multiple mRNAs using digoxigenin and radioisotopic cRNA probes.

We present a simple, reliable method for simultaneous detection of two distinct mRNAs within the same tissue sample by double in situ hybridization histochemistry. Sections are hybridized with a cocktail of radiolabeled and digoxigenin-labeled cRNA probes. The digoxigenin-labeled probe is detected with an alkaline phosphatase (AP)-dependent chromogen reaction and then the radiolabeled probe is detected by conventional autoradiography. The sensitivities of the two detection methods are comparable and demonstrate the feasibility of using double in situ hybridization histochemistry to investigate the co-localization and co-regulation of mRNAs that are expressed at relatively low levels.

Animals↗

PubMed1993

Expression of the cysteine proteinase inhibitor cystatin C gene in rat heart: use of digoxigenin-labeled probes generated by polymerase chain reaction directly for in situ and northern blot hybridizations.

Cystatins represent a widely distributed superfamily of cysteine proteinase inhibitory proteins. We investigated the expression of the cystatin C gene, belonging to the family 2 of cystatins, in the hearts of female rats. Using a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) we have detected cystatin C mRNA in the ventricule and atrium, as well as in liver and submandibular gland. A digoxigenin-labeled cystatin C probe, generated by PCR, hybridized to a single mRNA species of about 700 nucleotides on Northern blots. Northern blot hybridizations established that neither an acute inflammation produced by injection of turpentine nor administration of the beta-adrenergic agonist isoproterenol had an effect on the level of cystatin C mRNA in the heart. In situ hybridizations with digoxigenin-labeled probe localized the expression of the cystatin C gene to cardiac muscle fibers but not to other cardiac cellular elements. Cystatin C may be released by cardiac muscle fibers under physiological and pathological conditions and may modify inflammatory and necrobiotic processes.

Animals↗

PubMed1993

An efficient method to detect calcitonin mRNA in normal and neoplastic rat C-cells (medullary thyroid carcinoma) by in situ hybridization using a digoxigenin-labeled synthetic oligodeoxyribonucleotide probe.

We report here an efficient and rapid method for the specific detection of calcitonin in tumor C-cells of medullary thyroid carcinoma (MTC). This occasionally aggressive tumor arises from the endocrine thyroid C-cells. Its principal marker is calcitonin, the predominant C-cell secretion, which is detected in patients and in our animal model by radioimmunoassay of the plasma, as well as by immunohistochemistry of thyroid tissues. Although calcitonin is easily detectable in normal C-cells, its content is greatly reduced in tumor cells owing to the disappearance of the secretory granules that store the mature peptide. This finding suggests cell dedifferentiation correlated with an increasing aggressivity of the tumor. We therefore developed a rapid detection of calcitonin mRNA by in situ hybridization on routine paraffin sections, using a synthetic oligodeoxyribonucleotide probe labeled with digoxigenin-dUTP. The reaction was detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase, and the enzyme catalyzed the appearance of a dark blue color. The signal was exclusively restricted to the normal, hyperplastic, and tumor C-cells. It was specific, as increasing concentrations of the unlabeled oligonucleotide led to progressive disappearance of the reaction. Its sensitivity was slightly diminished as compared with corresponding frozen sections, but the intensity of the signal was quite acceptable. High levels of calcitonin mRNA were found in all normal and hyperplastic C-cells. They were increased in most of the tumor MTC cells, which did not correlate with the amount of intracellular peptide stores but explained the abnormally high basal levels of circulating calcitonin of the tumor-bearing rats. ISH is therefore of greater value than ICC for an early anatomopathological detection of this tumor. Our data show that the tumor cells are not "dedifferentiated." They only lack the granular compartment storing the mature peptide before exocytosis, but CT biosynthesis and the rest of the secretory process seem to be complete. Our results suggest that factors expressed in malignant C-cells affect basic cell mechanisms involved in the storage of the mature calcitonin, rather than the expression of the CALC gene.

Animals↗

PubMed1993

Detection of nerve growth factor mRNA in rodent salivary glands with digoxigenin- and 33P-labeled oligonucleotides: effects of castration and sympathectomy.

Nerve growth factor (NGF) is a protein highly expressed in the male mouse submandibular gland. We have applied a non-radioactive in situ hybridization method using digoxigenin-labeled NGF oligonucleotides, and have found the highest amounts of NGF mRNA in the secretory striated ducts of the male mouse submandibular gland. Scattered strongly positive cells were found in male mouse sublingual glands. Weakly labeled cells were seen in female mouse and in male rat submandibular gland striated duct cells. Using 33P as an alternative to 32P and 35S, we demonstrated a 1.3 KB NGF mRNA in salivary glands of male mice by Northern blot hybridization. Using 33P we detected NGF mRNA in male mouse submandibular glands by in situ hybridization but with a signal that, compared with the non-radioactive method, had a very low resolution. Castration of male mice almost abolished both the 1.3 KB NGF mRNA seen with Northern blots and the NGF mRNA labeling in submandibular glands 4 weeks after the operation, whereas levels were increased 6 hr and 2 days after sympathectomy. We conclude that hybridization with digoxigenin-labeled NGF oligonucleotides is a good tool to study the expression and regulation of NGF mRNA in male mouse submandibular glands.

Animals↗

PubMed1995

In situ hybridization of parathyroid hormone-related protein in normal skin, skin tumors, and gynecological cancers using digoxigenin-labeled probes and antibody enhancement.

We describe a novel procedure for in situ hybridization that combines the use of digoxigenin-labeled oligonucleotide probes with an antibody enhancement step that can be performed on formalin-fixed, paraffin-embedded tissues. Addition of a second antibody enhances the visibility of parathyroid hormone-related protein (PTHrP) mRNA expression from barely to highly discernible and interpretable, with virtually no nonspecific background expression. This technique has allowed visualization of PTHrP mRNA in normal human skin and epithelium-derived tumors. PTHrP mRNA expression was confined to the basal and spinous keratinocyte layers of skin. There was strong hybridization in the spinous keratinocyte layer and a low level of hybridization in the basal layer. An extensive panel of positive and negative controls included poly d(T) probe to indicate total mRNA present in the sections. Squamous cell carcinomas and basal cell carcinomas of the skin, from pathology archives, were examined for the presence of PTHrP mRNA. The results reflected previous immunohistochemical studies, with every squamous cell carcinoma hybridizing strongly with the PTHrP probes. The basal cell carcinomas showed no expression of PTHrP mRNA, although the total mRNA signal was very strong. The localization of PTHrP mRNA in the tumors of the gynecological tract also reflected the immunohistochemical findings, with expression found in the squamous cell carcinomas but not in the adenocarcinomas. In situ hybridization with digoxigenin-labeled oligonucleotide probes and antibody enhancement has provided a sensitive, highly specific procedure for detection of PTHrP mRNA in tumors and normal tissue.

Antibodies, Monoclonal↗

PubMed1998

Cellular localization of insulin-like growth factor II mRNA in the human fetus and the placenta: detection with a digoxigenin-labeled cRNA probe and immunocytochemistry.

IGF-II plays a major role in the regulation of human fetal growth and development. However, more extensive information on the cellular sites of IGF-II synthesis in the fetus would provide more insight into its role in fetal organogenesis. Thus we have determined the sites of IGF-II synthesis in 18-26-wk gestation human fetal tissues using in situ hybridization with a digoxigenin-labeled cRNA probe to localize IGF-II mRNA in fetal liver, kidney, adrenal gland, cerebral cortex, costal cartilage, skeletal muscle, and lung, and in placental tissue. In human fetal tissues it has to date been impossible to clearly assign IGF-II mRNA to epithelial cells of entodermal origin. Besides their already known localization in cell matrix and a variety of mesodermal cell types, strong IGF-II mRNA-positive signals were detected in epithelial cells in the liver (hepatocytes), bronchial and bronchiolar epithelium, undifferentiated renal tubular epithelium, mature glomerular epithelium, pelvic urothelium, and adrenal epithelial cells of the zona persistens. To identify the cellular location of immunoreactive IGF-II, we also performed immunocytochemical studies in tissues of the same fetuses. Every tissue studied except the cerebral cortex contained immunoreactive cells; however, immunostaining was generally weaker than in situ hybridization signals. Our data show that the distribution of IGF-II in human fetal tissue is much more widespread than hitherto thought. A digoxigenin-labeled detection system for IGF-II is more capable of detecting the cellular expression pattern of IGF-II than radioactive probes and is suitable for analysis of routinely prepared paraffin-embedded material.

Abortion, Induced↗

PubMed1998

Immunological detection and quantification of oxidized proteins by labelling with digoxigenin.

An immunological assay, based on the digoxigenin/anti-digoxigenin system, was developed to detect and quantify carbonyl moieties that result from oxidative damage to proteins. Bovine serum albumin (BSA) was oxidized by a hydroxyl radical-generating system consisting of ascorbate/Fe(III)/O2. The resulting albumin-derived carbonyls were labelled with digoxigenin-hydrazide and detected by dot blotting with an anti-digoxigenin antibody conjugated to alkaline phosphatase. Quantification was carried out by a densitometric analysis. This system allows the detection of a pmole-amount of carbonyl groups on blots. The assay covers a range of sensitivity from 1.26 to 126 pmoles. Another feature of this method is its application to a complex protein mixture (homogenate) to analyze the oxidative status of individual proteins, as are shown for intestinal brush border membrane homogenate of a rat.

Animals↗

PubMed1997

Digoxigenin-labeled peptides for the immunological quantification of intracellular signaling proteins: application to the MAP kinase kinase isoform MEK2.

Two competitive enzyme immunoassays using digoxigenin-labeled peptides have been developed for the quantification of the protein kinase MEK2 in cell extracts. Rabbit polyclonal antibodies directed against either the amino-terminal or proline-rich amino acid sequences of MEK2 were used for the immunoconcentration of the protein. Anti-digoxigenin Fab fragments labeled with horseradish peroxidase allowed the detection of the immune complexes. Amino-terminal and proline-rich enzyme immunoassays exhibited a sensitivity level of 63 and 71 fmol/mL, respectively, and displayed a half-maximal saturation value of 1320 and 1780 fmol/mL. The intra- and inter-assay coefficients of variation for both assays assessed at three different concentrations of MEK2 were lower than 6% and 12%, respectively. The amount of MEK2 measured by the two methods demonstrated an excellent correlation with the expression level of the protein detected by immunoblot analyses when tested on different cell lysates.

Animals↗

PubMed1993

Synthesis of digoxigenin-labeled cRNA probes for nonisotopic in situ hybridization using reverse transcription polymerase chain reaction.

Nonisotopic methods of mRNA in situ hybridization have distinct advantages over isotopic techniques. Nonisotopically labeled probes are stable and nontoxic, have short detection times, demonstrate excellent spatial resolution of their signals and have sensitivities comparable to radiolabeled probes. We developed a simple method of generating nonisotopically labeled cRNA probes which is based on the reverse transcription polymerase chain reaction (RT-PCR) and used it to synthesize a panel of probes for various murine extracellular matrix genes. Engelbreth-Holm-Swarm (EHS) tumor RNA was reverse transcribed and PCR was used to amplify defined regions of multiple extracellular matrix protein genes from the resulting first strand cDNAs. Bacteriophage promoters which had been incorporated into the PCR products were then used to generate digoxigenin-conjugated antisense and sense cRNAs. The antisense probes were employed to detect the specific extracellular matrix protein mRNAs in the EHS tumor by in situ hybridization. This technique provides a rapid and efficient alternative to conventional transcription systems which use plasmid vectors for the synthesis of digoxigenin-labeled cRNA probes.

Animals↗

PubMed2001

Expression of exon 13 from the Ki-67 gene in human cells and tissues by digoxigenin-labelled mRNA in situ hybridization.

OBJECTIVE: To get insight on the regulatory mechanism of Ki-67 gene expression in malignant cell cycle. METHODS: Non-radioactive in situ hybridization (ISH) was undertaken, combined with immunohistochemistry to study the Ki-67 gene transcription and translation in various human cells and tissues. HeLa cells and fresh colon cancer cells, tonsil, normal pancreas and pancreatic cancer tissues were used in this study. A 435 bp cDNA fragment located in exon 13 of the Ki-67 antigen gene was amplified by polymerase chain reaction (PCR). Digoxigenin-labelled antisense and sense RNA probes were prepared for detecting Ki-67 mRNA, combined with MIB-1 immunohistochemistry. RESULTS: Successful localization of Ki-67 mRNA in human HeLa cells, colon cancer cells, tissues specimen of the tonsil and pancreatic cancer tissue sections was accomplished by digoxigenin-labelling in situ hybridization technique. ISH to colon cancer cells and pancreatic cancer tissue slides showed that much stronger cytoplasm and perinuclear mRNA signals of the Ki-67 gene were present in malignant cells than in normal cells, which was in accordance with MIB-1 nuclear protein signals. CONCLUSIONS: A sensitive and practical in situ hybridization method for the analysis of Ki-67 antigen mRNA in human cell and tissue was developed. Abnormal transcription of exon 13 of Ki-67 gene might be responsible for malignant cell proliferation in colon and pancreatic cancer.

Digoxigenin↗