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Sample records for situ hybridization analyses

  1. Fluorescent in situ hybridization analyses of human oocytes in trisomy 18 and 21

    SciTech Connect

    Cheng, E.Y.; Chen, Y.J.; Gartler, S.M.

    1994-09-01

    The commonly accepted view of synapsis is that only 2 homologues can synapse at any one site and that this restriction applies to polyploids as well. However, triple synapsis has been observed is some triploid plants and in triploid chicken. In humans, triple synapsis of the long arm of chromosome 21 was detected in sperm of a trisomic 21 individual. More recently, studies of oocytes from trisomic 21 and 18 fetuses also indicated extensive triple synapsis along the entire length of the chromosomes. To further investigate this question, we undertook an evaluation of trivalent synapsis in fetal oocytes from 2 trisomic 21 and 2 trisomic 18 fetuses using fluorescent in situ hybridization (FISH) with whole chromosome probes. Oocytes were hybridized with whole chromosome probes obtained from ONCOR, Inc. after fixation with methanol and acetic acid. Slides were scored for the distribution of prophase stages, hybridization efficiency, and hybridization characteristics of chromosomes 18 and 21 in the trisomic 18 and 21 fetuses respectively. Fifty-eight per cent (379/650) of pachytenes analyzed for chromosome 18 contained a conspicous trivalent and 319 (48%) of these nuclei contained a single, thick, continuous fluorescent signal consistent with complete triple synapsis along the entire length of all 3 chromosomes. Sixteen per cent (104/650) of pachytene contained 2 signals consistent with a bivalent and a univalent, and 9 cells contained 3 thin signals consistent with asynapsis of all 3 chromosomes. The remaining 158 pachytenes had unusual pairing configurations that we could not classify, but they most likely represent trivalents with partial pairing between different homologues. In the 2 trisomic 21 fetuses, the majority (143/232) of pachytenes also contained one signal while only 52 cells contained a bivalent and univalent. Five cells contained 3 separate signals. These results confirm the existence of triple synapsis in human meiosis.

  2. Normal development of the tomato clownfish Amphiprion frenatus: live imaging and in situ hybridization analyses of mesodermal and neurectodermal development.

    PubMed

    Ghosh, J; Wilson, R W; Kudoh, T

    2009-12-01

    The normal embryonic development of the tomato clownfish Amphiprion frenatus was analysed using live imaging and by in situ hybridization for detection of mesodermal and neurectodermal development. Both morphology of live embryos and tissue-specific staining revealed significant differences in the gross developmental programme of A. frenatus compared with better-known teleost fish models, in particular, initiation of somitogenesis before complete epiboly, initiation of narrowing of the neurectoderm (neurulation) before somitogenesis, relatively early pigmentation of melanophores at the 10-15 somite stage and a distinctive pattern of melanophore distribution. These results suggest evolutionary adaptability of the teleost developmental programme. The ease of obtaining eggs, in vitro culture of the embryo, in situ staining analyses and these reported characteristics make A. frenatus a potentially important model marine fish species for studying embryonic development, physiology, ecology and evolution. PMID:20738687

  3. Metallographic in situ hybridization.

    PubMed

    Powell, Richard D; Pettay, James D; Powell, William C; Roche, Patrick C; Grogan, Thomas M; Hainfeld, James F; Tubbs, Raymond R

    2007-08-01

    Metallographic methods, in which a target is visualized using a probe or antibody that deposits metal selectively at its binding site, offers many advantages for bright-field in situ hybridization (ISH) detection as well as for other labeling and detection methods. Autometallographically enhanced gold labeling procedures have demonstrated higher sensitivity than conventional enzyme chromogens. Enzyme metallography, a novel procedure in which an enzymatic probe is used to deposit metal directly from solution, has been used to develop bright-field ISH methods for HER2 gene determination in breast cancer and other biopsy specimens. It provides the highest level of sensitivity and resolution, both for visualizing endogenous gene copies in nonamplified tissues and for resolving multiple gene copies to allow copy enumeration in amplified tissues without the need for oil immersion or fluorescence optics. An automated enzyme metallography procedure, silver ISH, has been developed for use in slide-staining instruments. Metallographic staining also provides excellent results for immunohistochemistry and may be combined with other staining procedures for the simultaneous detection of more than one gene or combinations of genes and proteins. PMID:17640553

  4. Triplex in-situ hybridization

    DOEpatents

    Fresco, Jacques R.; Johnson, Marion D.

    2002-01-01

    Disclosed are methods for detecting in situ the presence of a target sequence in a substantially double-stranded nucleic acid segment, which comprises: a) contacting in situ under conditions suitable for hybridization a substantially double-stranded nucleic acid segment with a detectable third strand, said third strand being capable of hybridizing to at least a portion of the target sequence to form a triple-stranded structure, if said target sequence is present; and b) detecting whether hybridization between the third strand and the target sequence has occured.

  5. In situ hybridization in the plant Kalanchoë daigremontiana.

    PubMed

    Garcês, Helena; Sinha, Neelima

    2009-10-01

    Here we describe in detail the detection of gene expression in plant tissues of Kalanchoë daigremontiana by in situ hybridization analyses. Included are methods for making RNA transcript probes, probe-tissue hybridization, and detection of antisense RNA probes. The in situ hybridization technique is used to determine which cells or group of cells in particular tissue(s) express a gene of interest. PMID:20147047

  6. Reutilization of previously hybridized slides for fluorescence in situ hybridization

    SciTech Connect

    Epstein, L.; DeVries, S.; Waldman, F.M.

    1995-12-01

    Application of fluorescence in situ hybridization (FISH) to clinical material is sometimes limited by sample size. In addition, heterogeneity among slides prepared from a single sample may lead to variation in FISH analyses. Reutilization of material for repeated FISH analyses would help to alleviate these problems. We have developed a simple procedure for repeated FISH analyses with directly conjugated probes. Previously hybridized probes are removed by incubation in denaturing solution, and slides can then be rehybridized without residual signals remaining. Several cycles of this procedure allow a full complement of chromosomal loci to be analyzed on the same population of cells. Advantages of this protocol include gaining more cytogenetic information from small samples and eliminating the problem of intratumorvariability. 5 refs., 4 figs.

  7. Painting of parental chromatin in Beta hybrids by multi-colour fluorescent in situ hybridization.

    PubMed

    Desel, Christine; Jansen, Rita; Dedong, Gue; Schmidt, Thomas

    2002-02-01

    Sugar beet (Beta vulgaris L.) is a relatively young crop and has a narrow gene pool. In order to introduce genetic variability into the crop, interspecific hybrids, selected from crosses with wild beets of the sections Corollinae and Procumbentes, have been generated. The introgressed B. procumbens chromatin carries resistance genes to beet cyst nematode Heterodera schachtii Schm. These lines are important for breeding of nematode-resistant sugar beet, while Corollinae species are potential donors of tolerance to biotic and abiotic stresses such as drought or saline soils. We have used in situ hybridization of genomic DNA to discriminate the parental chromosomes in these interspecific hybrids. Suppression of cross-hybridization by blocking DNA was not necessary indicating that the investigated Beta genomes contain sufficient species-specific DNA enabling the unequivocal determination of the genomic composition of the hybrids. Interspecific hybrid lines with an additional chromosome (2n = 18 + 1), chromosome fragment (2n = 18 + fragment) or translocation of B. procumbens (2n = 18) were analysed by genomic in situ hybridization (GISH) at mitosis and meiosis. Species-specific satellites and ribosomal genes used in combination with genomic DNA or in rehybridization experiments served as landmark probes for chromosome identification in hybrid genomes. The detection of a B. procumbens translocation of approx. I Mbp demonstrated the sensitivity and resolution of GISH and showed that this approach is a powerful method in genome analysis projects of the genus Beta. PMID:12099348

  8. DNA/DNA in situ hybridization with enzyme linked probes

    SciTech Connect

    Grillo, S.; Mosher, M.; Charles, P.; Henry, S.; Taub, F.

    1987-05-01

    A non-radioactive in situ nucleic acid hybridization method which requires no antibodies, haptens, avidin or biotin intermediateries is presented. Horseradish peroxidase (HRP) labeled nucleic acid probes are hybridized in situ for 2 hours or less, followed by brief washing of hybridized cells and the direct detection of in situ hybrids with diaminobenzidine (DAB). Application of this method to the detection of Human Papilloma Virus (HPV) in human cells is shown.

  9. Zebrafish Whole-Mount In Situ Hybridization Followed by Sectioning.

    PubMed

    Doganli, Canan; Nyengaard, Jens Randel; Lykke-Hartmann, Karin

    2016-01-01

    In situ hybridization is a powerful technique used for locating specific nucleic acid targets within morphologically preserved tissues and cell preparations. A labeled RNA or DNA probe hybridizes to its complementary mRNA or DNA sequence within a sample. Here, we describe RNA in situ hybridization protocol for whole-mount zebrafish embryos. PMID:26695046

  10. Molecular cytogenetics using fluorescence in situ hybridization

    SciTech Connect

    Gray, J.W.; Kuo, Wen-Lin; Lucas, J.; Pinkel, D.; Weier, H-U.; Yu, Loh-Chung.

    1990-12-07

    Fluorescence in situ hybridization (FISH) with chromosome-specific probes enables several new areas of cytogenetic investigation by allowing visual determination of the presence and normality of specific genetic sequences in single metaphase or interphase cells. in this approach, termed molecular cytogenetics, the genetic loci to be analyzed are made microscopically visible in single cells using in situ hybridization with nucleic acid probes specific to these loci. To accomplish this, the DNA in the target cells is made single stranded by thermal denaturation and incubated with single-stranded, chemically modified probe under conditions where the probe will anneal only with DNA sequences to which it has high DNA sequence homology. The bound probe is then made visible by treatment with a fluorescent reagent such as fluorescein that binds to the chemical modification carried by the probe. The DNA to which the probe does not bind is made visible by staining with a dye such as propidium iodide that fluoresces at a wavelength different from that of the reagent used for probe visualization. We show in this report that probes are now available that make this technique useful for biological dosimetry, prenatal diagnosis and cancer biology. 31 refs., 3 figs.

  11. Painting of Parental Chromatin in Beta Hybrids by Multi‐colour Fluorescent in situ Hybridization

    PubMed Central

    DESEL, CHRISTINE; JANSEN, RITA; DEDONG, GUE; SCHMIDT, THOMAS

    2002-01-01

    Sugar beet (Beta vulgaris L.) is a relatively young crop and has a narrow gene pool. In order to introduce genetic variability into the crop, interspecific hybrids, selected from crosses with wild beets of the sections Corollinae and Procumbentes, have been generated. The introgressed B. procumbens chromatin carries resistance genes to beet cyst nematode Heterodera schachtii Schm. These lines are important for breeding of nematode‐resistant sugar beet, while Corollinae species are potential donors of tolerance to biotic and abiotic stresses such as drought or saline soils. We have used in situ hybridization of genomic DNA to discriminate the parental chromosomes in these interspecific hybrids. Suppression of cross‐hybridization by blocking DNA was not necessary indicating that the investigated Beta genomes contain sufficient species‐specific DNA enabling the unequivocal determination of the genomic composition of the hybrids. Interspecific hybrid lines with an additional chromosome (2n = 18 + 1), chromosome fragment (2n = 18 + fragment) or translocation of B. procumbens (2n = 18) were analysed by genomic in situ hybridization (GISH) at mitosis and meiosis. Species‐specific satellites and ribosomal genes used in combination with genomic DNA or in rehybridization experiments served as landmark probes for chromosome identification in hybrid genomes. The detection of a B. procumbens translocation of approx. 1 Mbp demonstrated the sensitivity and resolution of GISH and showed that this approach is a powerful method in genome analysis projects of the genus Beta. PMID:12099348

  12. Autofluorescence correction for fluorescence in situ hybridization

    SciTech Connect

    Szoelloesi, J.; Balazs, M.; Waldman, F.C.

    1995-08-01

    Optimal sensitivity of fluorescence in situ hybridization (FISH) requires bright signals and low background fluorescence. Use of locus-specific probes is especially dependent on high sensitivity. Some tissue preparations show high autofluorescence, masking small or dim signals. We have developed a new method for subtracting autofluorescence from digital images on a pixel-by-pixel basis. It is based on the observation that fluorescent labels for FISH have narrower excitation and emission spectra than the chemical components responsible for autofluorescence. Our new approach uses calculation of the ratio of autofluorescence between multiple color images for correction of autofluorescence in each individual image. By subtracting autofluorescence components, we were able to enhance centromeric signals and make previously indistiguishable cosmid signals clearly visible. This image-processing approach to autofluorescence correction may widen the applicability of gene-specific probes in FISH analysis of tumor material. 15 refs., 3 fig., 1 tab.

  13. In situ hybridization for detection of HIV RNA.

    PubMed

    Fox, C H; Cottler-Fox, M

    2001-05-01

    In HIV studies, in situ hybridization can be used for identifying virion RNA, mRNA being produced for virion packaging, and proviral DNA in the cytoplasm or integrated in the nucleus. This unit focuses primarily on identifying virion RNA, because this is the most sensitive means by which in situ hybridization can be employed to detect HIV expression. In situ hybridization, as developed for HIV RNA detection, involves several protocols: (1) preparation of a radioactive or nonradioactive RNA probe; (2) in situ hybridization of probe to cells and paraffin sections of tissue; (3) detection of radiolabeled probe by emulsion autoradiography; (4) development, staining, and mounting of slides; and finally (5) examination of slides by bright-field, dark-field, specular reflectance, or laser-scanning confocal microscopy. The protocols presented in this unit describe a setup involving up to 150 slides. PMID:18432712

  14. Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization.

    PubMed

    Wienberg, J; Jauch, A; Stanyon, R; Cremer, T

    1990-10-01

    A new strategy for analyzing chromosomal evolution in primates is presented using chromosomal in situ suppression (CISS) hybridization. Biotin-labeled DNA libraries from flow-sorted human chromosomes are hybridized to chromosome preparations of catarrhines, platyrrhines, and prosimians. By this approach rearrangements of chromosomes that occurred during hominoid evolution are visualized directly at the level of DNA sequences, even in primate species with pronounced chromosomal shuffles. PMID:2249853

  15. In situ hybridization for metalloproteinases and their inhibitors.

    PubMed

    Hurskainen, Tiina L; Apte, Suneel S

    2010-01-01

    In situ hybridization (ISH) is an invaluable tool in understanding tissue-specific gene expression and gene regulation within a spatial context and at a resolution that is not possible by any other method. In this chapter, we provide ISH methodology that has successfully been applied to the detection of metalloproteinases and their inhibitors. PMID:20135283

  16. FISH-ing for Genes: Modeling Fluorescence "in situ" Hybridization

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton Buck

    2006-01-01

    Teaching methods of genetic analysis such as fluorescence in situ hybridization (FISH) can be an important part of instructional units in biology, microbiology, and biotechnology. Experience, however, indicates that these topics are difficult for many students. The authors of this article describe how they created an activity that effectively…

  17. Supernumerary ring chromosome 17 identified by fluorescent in situ hybridization

    SciTech Connect

    Fagan, K.; Edwards, M.

    1997-04-14

    We present a patient with multiple anomalies and severe developmental delay. A small supernumerary ring chromosome was found in 40% of her lymphocyte cells at birth. The origin of the marker chromosome could not be determined by GTG banding, but fluorescent in situ hybridization (FISH) later identified the marker as deriving from chromosome 17. 20 refs., 2 figs., 1 tab.

  18. Strategies for In situ and Sample Return Analyses

    NASA Astrophysics Data System (ADS)

    Papanastassiou, D. A.

    2006-12-01

    There is general agreement that planetary exploration proceeds from orbital reconnaissance of a planet, to surface and near-surface in situ exploration, to sample return missions, which bring back samples for investigations in terrestrial laboratories, using the panoply of state-of-the-art analytical techniques. The applicable techniques may depend on the nature of the returned material and complementary and multi- disciplinary techniques can be used to best advantage. High precision techniques also serve to provide the "ground truth" and calibrate past and future orbital and in situ measurements on a planet. It is also recognized that returned samples may continue to be analyzed by novel techniques as the techniques become developed, in part to address specific characteristics of returned samples. There are geophysical measurements such as those of the moment of inertia of a planet, seismic activity, and surface morphology that depend on orbital and in-situ science. Other characteristics, such as isotopic ages and isotopic compositions (e.g., initial Sr and Nd) as indicators of planetary mantle or crust evolution and sample provenance require returned samples. In situ analyses may be useful for preliminary characterization and for optimization of sample selection for sample return. In situ analyses by Surveyor on the Moon helped identify the major element chemistry of lunar samples and the need for high precision mass spectrometry (e. g., for Rb-Sr ages, based on extremely low alkali contents). The discussion of in-situ investigations vs. investigations on returned samples must be directly related to available instrumentation and to instrumentation that can be developed in the foreseeable future. The discussion of choices is not a philosophical but instead a very practical issue: what precision is required for key investigations and what is the instrumentation that meets or exceeds the required precision. This must be applied to potential in situ instruments and

  19. In situ nanofabrication of hybrid PEG-dendritic-inorganic nanoparticles and preliminary evaluation of their biocompatibility.

    PubMed

    Sousa-Herves, Ana; Sánchez Espinel, Christian; Fahmi, Amir; González-Fernández, África; Fernandez-Megia, Eduardo

    2015-03-01

    An in situ template fabrication of inorganic nanoparticles using carboxylated PEG-dendritic block copolymers of the GATG family is described as a function of the dendritic block generation, the metal (Au, CdSe) and metal molar ratio. The biocompatibility of the generated nanoparticles analysed in terms of their aggregation in physiological media, cytotoxicity and uptake by macrophages relates to the PEG density of the surface of the hybrids. PMID:25530028

  20. Nucleic acid in-situ hybridization detection of infectious agents

    NASA Astrophysics Data System (ADS)

    Thompson, Curtis T.

    2000-04-01

    Limitations of traditional culture methods and newer polymerase chain reaction (PCR)-based methods for detection and speciation of infectious agents demonstrate the need for more rapid and better diagnostics. Nucleic acid hybridization is a detection technology that has gained wide acceptance in cancer and prenatal cytogenetics. Using a modification of the nucleic acid hybridization technique known as fluorescence in-situ hybridization, infectious agents can be detected in a variety of specimens with high sensitivity and specificity. The specimens derive from all types of human and animal sources including body fluids, tissue aspirates and biopsy material. Nucleic acid hybridization can be performed in less than one hour. The result can be interpreted either using traditional fluorescence microscopy or automated platforms such as micro arrays. This paper demonstrates proof of concept for nucleic acid hybridization detection of different infectious agents. Interpretation within a cytologic and histologic context is possible with fluorescence microscopic analysis, thereby providing confirmatory evidence of hybridization. With careful probe selection, nucleic acid hybridization promises to be a highly sensitive and specific practical diagnostic alternative to culture, traditional staining methods, immunohistochemistry and complicated nucleic acid amplification tests.

  1. Hybrid Propulsion In-Situ Resource Utilization Test Facility Development

    NASA Technical Reports Server (NTRS)

    Chandler, Ashley A.; Gatto, Corinne; Nakazono, Barry; Grayson, Kristian; Vaughan, David

    2014-01-01

    Hybrid propulsion could be a potential game changing technology for several Mars applications, such as Mars Sample Return (MSR) and human exploration. A flexible hybrid test facility has been built at the Jet Propulsion Laboratory to provide data relevant to the design of such systems. This paper presents the motivations for such a system and its design. The facility is capable of testing 5 cm diameter fuel grains with gaseous oxygen and Mars in situ propellant production simulating oxidizer (varying mixtures of GO2, CO2 and CO). All currently planned tests utilize paraffin based fuels; however, alternative hybrid fuels may be used in the future. Variable length to outer diameter (L/D) ratios may also be tested to give insight on potential packaging constraints. The goal of this research is to enable the inclusion of hybrid propulsion systems in future mission design studies by determining the empirical constants in the regression rate equation for paraffin-based fuels with space storable and/or in situ oxidizers and to investigate the effect of L/D on combustion efficiency. Test results will be reported separately.

  2. Fluorescent In Situ Hybridization in Suspension by Imaging Flow Cytometry.

    PubMed

    Maguire, Orla; Wallace, Paul K; Minderman, Hans

    2016-01-01

    The emergence of imaging flow cytometry (IFC) has brought novel applications exploiting its advantages over conventional flow cytometry and microscopy. One of the new applications is fluorescence in situ hybridization in suspension (FISH-IS). Conventional FISH is a slide-based approach in which the spotlike imagery resulting from hybridization with fluorescently tagged probes is evaluated by fluorescence microscopy. The FISH-IS approach evaluated by IFC enables the evaluation of tens to hundreds of thousands of cells in suspension and the analysis can be automated and standardized diminishing operator bias from the analysis. The high cell number throughput of FISH-IS improves the detection of rare events compared to conventional FISH. The applicability of FISH-IS is currently limited to detection of abnormal quantitative differences of hybridization targets such as occur in numerical chromosome abnormalities, deletions and amplifications.Here, we describe a protocol for FISH-IS using chromosome enumeration probes as an example. PMID:27460240

  3. Identification of mosaicism in Prader-Willi syndrome using fluorescent in situ hybridization

    SciTech Connect

    Mowery-Rushton, P.A.; Surti, U.; Hanchett, J.M.

    1996-12-30

    We report on our findings of 4 patients with mosaicism for a deletion of chromosome 15, most commonly associated with Prader-Willi syndrome (PWS). We examined a series of typical and atypical PWS patients in order to identify cytogenetically undetected deletions, using fluorescence in situ hybridization. In 4 of the patients analyzed we detected a deletion in 14-60% of peripheral blood leukocytes, using four commercially available probes. Our results indicate that mosaicism may play a role in the etiology of some PWS cases. These findings may be especially useful in patients who display discrepancies between clinical phenotype and established diagnostic criteria. Methylation and microsatellite polymorphism analyses of 2 patients with low-level mosaicism failed to identify the deletion. We propose that fluorescence in situ hybridization is the most effective method for detecting somatic mosaicism, since a large number of cells can be individually examined for the presence or absence of a specific deletion. 47 refs., 5 figs., 3 tabs.

  4. Genetic profiling of yeast industrial strains using in situ comparative genomic hybridization (CGH).

    PubMed

    Wnuk, Maciej; Panek, Anita; Golec, Ewelina; Magda, Michal; Deregowska, Anna; Adamczyk, Jagoda; Lewinska, Anna

    2015-09-20

    The genetic differences and changes in genomic stability may affect fermentation processes involving baker's, brewer's and wine yeast strains. Thus, it seems worthwhile to monitor the changes in genomic DNA copy number of industrial strains. In the present study, we developed an in situ comparative genomic hybridization (CGH) to investigate the ploidy and genetic differences between selected industrial yeast strains. The CGH-based system was validated using the laboratory Saccharomyces cerevisiae yeast strains (haploid BY4741 and diploid BY4743). DNA isolated from BY4743 cells was considered a reference DNA. The ploidy and DNA gains and losses of baker's, brewer's and wine strains were revealed. Taken together, the in situ CGH was shown a helpful molecular tool to identify genomic differences between yeast industrial strains. Moreover, the in situ CGH-based system may be used at the single-cell level of analysis to supplement array-based techniques and high-throughput analyses at the population scale. PMID:26116136

  5. Telomere analysis by fluorescence in situ hybridization and flow cytometry.

    PubMed Central

    Hultdin, M; Grönlund, E; Norrback, K; Eriksson-Lindström, E; Just, T; Roos, G

    1998-01-01

    Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase. PMID:9685479

  6. EBER in situ hybridization for Epstein-Barr virus.

    PubMed

    Weiss, Lawrence M; Chen, Yuan-Yuan

    2013-01-01

    Epstein-Barr encoding region (EBER) in situ hybridization is the methodology of choice for the detection of the Epstein-Barr virus (EBV) in tissue sections. Because of the large numbers of copies of EBERs present in latently infected cells, non-isotopic methods can be used. Positive studies show staining in the nuclei of the EBV-infected cells, accentuating the chromatin and often excluding the nucleolus. False-negative results are most often the result of RNA degradation in the tissues, a finding that may be detected through the use of a polyT probe as a control for RNA preservation. PMID:23666702

  7. 10p Duplication characterized by fluorescence in situ hybridization

    SciTech Connect

    Wiktor, A.; Feldman, G.L.; Van Dyke, D.L.; Kratkoczki, P.; Ditmars, D.M. Jr.

    1994-09-01

    We describe a patient with severe failure to thrive, mild-moderate developmental delay, cleft lip and palate, and other anomalies. Routine cytogenetic analysis documented a de novo chromosome rearrangement involving chromosome 4, but the origin of the derived material was unknown. Using chromosome specific painting probes, the karyotype was defined as 46,XY,der(4)t(4;10)(q35;p11.23). Characterization of the dup(10p) by fluorescence in situ hybridization (FISH) analysis provides another example of the usefulness of this technology in identifying small deletions, duplications, or supernumerary marker chromosomes. 19 refs., 4 figs.

  8. Hybrid-type temperature sensor for in situ measurement

    SciTech Connect

    Iuchi, Tohru; Hiraka, Kensuke

    2006-11-15

    A hybrid-type surface temperature sensor combines the contact and noncontact methods, which allows us to overcome the shortcomings of both methods. The hybrid-type surface thermometer is composed mainly of two components: a metal film sheet that makes contact with an object and a radiometer that is used to detect the radiance of the rear surface of the metal film, which is actually a modified radiation thermometer. Temperature measurement using the hybrid-type thermometer with a several tens micrometer thick Hastelloy sheet, a highly heat and corrosion resistant alloy, is possible with a systematic error of -0.5 K and random errors of {+-}0.5 K, in the temperature range from 900 to 1000 K. This thermometer provides a useful means for calibration of in situ temperature measurement in various processes, especially in the silicon semiconductor industry. This article introduces the basic idea of the hybrid-type surface sensor, presents experimental results and discussions, and finally describes some applications.

  9. Bacterial colonization of enamel in situ investigated using fluorescence in situ hybridization.

    PubMed

    Al-Ahmad, Ali; Follo, Marie; Selzer, Ann-Carina; Hellwig, Elmar; Hannig, Matthias; Hannig, Christian

    2009-10-01

    Oral biofilms are one of the greatest challenges in dental research. The present study aimed to investigate initial bacterial colonization of enamel surfaces in situ using fluorescence in situ hybridization (FISH) over a 12 h period. For this purpose, bovine enamel slabs were fixed on buccal sites of individual splints worn by six subjects for 2, 6 and 12 h to allow biofilm formation. Specimens were processed for FISH and evaluated with confocal laser-scanning microscopy, using probes for eubacteria, Streptococcus species, Veillonella species, Fusobacterium nucleatum and Actinomyces naeslundii. The number of adherent bacteria increased with time and all tested bacterial species were detected in the biofilm formed in situ. The general percentage composition of the eubacteria did not change over the investigated period, but the number of streptococci, the most frequently detected species, increased significantly with time (2 h: 17.7+/-13.8 %; 6 h: 20.0+/-16.6 %; 12 h: 24.7+/-16.1 %). However, < or =1 % of the surface was covered with bacteria after 12 h of biofilm formation in situ. In conclusion, FISH is an appropriate method for quantifying initial biofilm formation in situ, and the proportion of streptococci increases during the first 12 h of bacterial adherence. PMID:19528150

  10. RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization

    PubMed Central

    Moffitt, Jeffrey R.; Zhuang, Xiaowei

    2016-01-01

    Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves. PMID:27241748

  11. The Use of Whole-Mount "in Situ" Hybridization to Illustrate Gene Expression Regulation

    ERIC Educational Resources Information Center

    Llamusí, Beatriz; Muñoz-Soriano, Verónica; Paricio, Nuria; Artero, Rubén

    2014-01-01

    "In situ" hybridization is a widely used technique for studying gene expression. Here, we describe two experiments addressed to postgraduate genetics students in which the effect of transcription factors on gene expression is analyzed in "Drosophila embryos of different genotypes by whole-mount in situ hybridization. In one of the…

  12. Human cDNA mapping using fluorescence in situ hybridization

    SciTech Connect

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  13. Detection of viral RNA by fluorescence in situ hybridization (FISH).

    PubMed

    Vyboh, Kishanda; Ajamian, Lara; Mouland, Andrew J

    2012-01-01

    localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev), FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus. Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF. PMID:22588480

  14. In situ nanofabrication of hybrid PEG-dendritic-inorganic nanoparticles and preliminary evaluation of their biocompatibility

    NASA Astrophysics Data System (ADS)

    Sousa-Herves, Ana; Sánchez Espinel, Christian; Fahmi, Amir; González-Fernández, África; Fernandez-Megia, Eduardo

    2015-02-01

    An in situ template fabrication of inorganic nanoparticles using carboxylated PEG-dendritic block copolymers of the GATG family is described as a function of the dendritic block generation, the metal (Au, CdSe) and metal molar ratio. The biocompatibility of the generated nanoparticles analysed in terms of their aggregation in physiological media, cytotoxicity and uptake by macrophages relates to the PEG density of the surface of the hybrids.An in situ template fabrication of inorganic nanoparticles using carboxylated PEG-dendritic block copolymers of the GATG family is described as a function of the dendritic block generation, the metal (Au, CdSe) and metal molar ratio. The biocompatibility of the generated nanoparticles analysed in terms of their aggregation in physiological media, cytotoxicity and uptake by macrophages relates to the PEG density of the surface of the hybrids. Electronic supplementary information (ESI) available: Structure of carboxylated PEG-GATG copolymers, aggregation of CdSe NPs in serum, and cytotoxicity of PEG-GATG copolymers. See DOI: 10.1039/c4nr06155a

  15. Small RNA Detection by in Situ Hybridization Methods

    PubMed Central

    Urbanek, Martyna O.; Nawrocka, Anna U.; Krzyzosiak, Wlodzimierz J.

    2015-01-01

    Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics. PMID:26068454

  16. Small RNA Detection by in Situ Hybridization Methods.

    PubMed

    Urbanek, Martyna O; Nawrocka, Anna U; Krzyzosiak, Wlodzimierz J

    2015-01-01

    Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics. PMID:26068454

  17. Improved hybrid solar cells via in situ UV-polymerization.

    SciTech Connect

    Tepavcevic, S.; Darling, S. B.; Dimitrijevic, N. M.; Rajh, T.; Sibener, S. J.; Univ. of Chicago

    2009-08-03

    One approach for making inexpensive inorganic-organic hybrid photovoltaic (PV) cells is to fill highly ordered TiO{sub 2} nanotube (NT) arrays with solid organic hole conductors such as conjugated polymers. Here, a new in situ UV polymerization method for growing polythiophene (UV-PT) inside TiO{sub 2} NTs is presented and compared to the conventional approach of infiltrating NTs with pre-synthesized polymer. A nanotubular TiO{sub 2} substrate is immersed in a 2,5-diiodothiophene (DIT) monomer precursor solution and then irradiated with UV light. The selective UV photodissociation of the C-I bond produces monomer radicals with intact {pi}-ring structure that further produce longer oligothiophene/PT molecules. Complete photoluminescence quenching upon UV irradiation suggests coupling between radicals created from DIT and at the TiO{sub 2} surface via a charge transfer complex. Coupling with the TiO{sub 2} surface improves UV-PT crystallinity and {pi}-{pi} stacking; flat photocurrent values show that charge recombination during hole transport through the polymer is negligible. A non-ideal, backside-illuminated setup under illumination of 620-nm light yields a photocurrent density of {approx} 5 {micro}A cm{sup -2} - surprisingly much stronger than with comparable devices fabricated with polymer synthesized ex situ. Since in this backside architecture setup we illuminate the cell through the Ag top electrode, there is a possibility for Ag plasmon-enhanced solar energy conversion. By using this simple in situ UV polymerization method that couples the conjugated polymer to the TiO{sub 2} surface, the absorption of sunlight can be improved and the charge carrier mobility of the photoactive layer can be enhanced.

  18. Improved hybrid solar cells via in situ UV polymerization.

    PubMed

    Tepavcevic, Sanja; Darling, Seth B; Dimitrijevic, Nada M; Rajh, Tijana; Sibener, Steven J

    2009-08-01

    One approach for making inexpensive inorganic-organic hybrid photovoltaic (PV) cells is to fill highly ordered TiO(2) nanotube (NT) arrays with solid organic hole conductors such as conjugated polymers. Here, a new in situ UV polymerization method for growing polythiophene (UV-PT) inside TiO(2) NTs is presented and compared to the conventional approach of infiltrating NTs with pre-synthesized polymer. A nanotubular TiO(2) substrate is immersed in a 2,5-diiodothiophene (DIT) monomer precursor solution and then irradiated with UV light. The selective UV photodissociation of the C--I bond produces monomer radicals with intact pi-ring structure that further produce longer oligothiophene/PT molecules. Complete photoluminescence quenching upon UV irradiation suggests coupling between radicals created from DIT and at the TiO(2) surface via a charge transfer complex. Coupling with the TiO(2) surface improves UV-PT crystallinity and pi-pi stacking; flat photocurrent values show that charge recombination during hole transport through the polymer is negligible. A non-ideal, backside-illuminated setup under illumination of 620-nm light yields a photocurrent density of approximately 5 microA cm(2)-surprisingly much stronger than with comparable devices fabricated with polymer synthesized ex situ. Since in this backside architecture setup we illuminate the cell through the Ag top electrode, there is a possibility for Ag plasmon-enhanced solar energy conversion. By using this simple in situ UV polymerization method that couples the conjugated polymer to the TiO(2) surface, the absorption of sunlight can be improved and the charge carrier mobility of the photoactive layer can be enhanced. PMID:19367599

  19. In situ hybridization of carbon nanotubes with bacterial cellulose for three-dimensional hybrid bioscaffolds.

    PubMed

    Park, Subeom; Park, Jooyeon; Jo, Insu; Cho, Sung-Pyo; Sung, Dongchul; Ryu, Seungmi; Park, Minsung; Min, Kyung-Ah; Kim, Jangho; Hong, Suklyun; Hong, Byung Hee; Kim, Byung-Soo

    2015-07-01

    Carbon nanotubes (CNTs) have shown great potential in biomedical fields. However, in vivo applications of CNTs for regenerative medicine have been hampered by difficulties associated with the fabrication of three-dimensional (3D) scaffolds of CNTs due to CNTs' nano-scale nature. In this study, we devised a new method for biosynthesis of CNT-based 3D scaffold by in situ hybridizing CNTs with bacterial cellulose (BC), which has a structure ideal for tissue-engineering scaffolds. This was achieved simply by culturing Gluconacetobacter xylinus, BC-synthesizing bacteria, in medium containing CNTs. However, pristine CNTs aggregated in medium, which hampers homogeneous hybridization of CNTs with BC scaffolds, and the binding energy between hydrophobic pristine CNTs and hydrophilic BC was too small for the hybridization to occur. To overcome these problems, an amphiphilic comb-like polymer (APCLP) was adsorbed on CNTs. Unlike CNT-coated BC scaffolds (CNT-BC-Imm) formed by immersing 3D BC scaffolds in CNT solution, the APCLP-adsorbed CNT-BC hybrid scaffold (CNT-BC-Syn) showed homogeneously distributed CNTs throughout the 3D microporous structure of BC. Importantly, in contrast to CNT-BC-Imm scaffolds, CNT-BC-Syn scaffolds showed excellent osteoconductivity and osteoinductivity that led to high bone regeneration efficacy. This strategy may open a new avenue for development of 3D biofunctional scaffolds for regenerative medicine. PMID:25941786

  20. Genomic In Situ Hybridization (GISH) as a Tool to Identify Chromosomes of Parental Species in Sunflower Interspecific Hybrids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interspecific hybridization has been widely used to transfer genes from wild species into cultivated sunflower. Fluorescent genomic in situ hybridization (GISH) has been used to identify alien chromosomes or segments in other crops, but an equivalent technique for sunflower is lacking. The objective...

  1. Applications of Strand-Specific in situ Hybridization

    SciTech Connect

    Goodwin, E.H.; Meyne, J.; Bailey, S.M.; Quigley, D.; Smith, L.; Tennyson, R.

    1997-01-01

    This is the final report of a three-year, Laboratory-Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Fluorescence in situ hybridization (FISH) is used to determine the location of specific DNA sequences on chromosomes. It is an effective tool in genomic mapping and is finding increasing use in medical diagnosis. A ''strand-specific'' version of FISH has been developed in the Life Sciences Division of LANL. The new procedure, named CO-FISH, reveals not only location but also the 5'-to-3'direction of a target sequence, such as the sense strand of a gene. This project was designed to investigate applications of the new technique. Strand-specific FISH was found to be useful and informative for genomic mapping of repetitive DNA sequences. The method provide a valuable new tool for investigating the mechanisms of aneuploidy inducing agents and the cytogenetic phenomena called lateral asymmetry. Finally, using strand-specific FISH, the authors were able to detect certain types of chromosome aberrations (isochromosomes, inversions and Robertsonian translocations) that can be difficult to observe with standard techniques.

  2. Regulatory pathway analysis by high-throughput in situ hybridization

    SciTech Connect

    Visel, Axel; Carson, James P.; Oldekamp, Judit; Warnecke, Marei; Jakubcakova, Vladimira; Zhou, Xunlei; Shaw, Chad; Alvarez-Bolado, Gonzalo; Eichele, Gregor

    2007-10-01

    Automated in situ hybridization (ISH) permits construction of comprehensive atlases of gene expression patterns in mammals. When web-accessible, such atlases become searchable digital expression maps of individual genes and offer an entryway to elucidate genetic interactions and signaling pathways. An atlas housing ~1,000 spatial gene expression patterns of the mid-gestation mouse embryo was generated. Patterns were textually annotated using a controlled vocabulary comprising 90 anatomical features. Hierarchical clustering of annotations was carried out using distance scores calculated from the similarity between pairs of patterns across all anatomical structures. This ordered hundreds of complex expression patterns into a matrix that reflected the embryonic architecture and the relatedness of patterns of expression. Clustering yielded twelve distinct groups of expression pattern. Because of similarity of expression patterns within a group, members of this group may be components of regulatory cascades. We focused on one group, which is composed of 83 genes, including Pax6, an evolutionary conserved transcriptional master mediator of the development. Using functional studies, ISH on Pax6-deficient embryos and Pax6 binding site identification and validation by means of electromobility shift assays, we identified numerous genes that are transcriptionally regulated by Pax6. Hence cluster analysis of annotated gene expression patterns obtained by robotic ISH is an entryway for identification of components of signaling cascades in mammals.

  3. Circovirus-infected geese studied by in situ hybridization.

    PubMed

    Smyth, Joan; Soike, D; Moffett, Deborah; Weston, J H; Todd, D

    2005-06-01

    It has now been established that circovirus infection is common in farmed geese, but little is known about the clinicopathological significance of such infections. Ten clinically diseased geese suspected of being infected by circovirus were studied by in situ hybridization using a goose circovirus DNA probe. Circovirus DNA was demonstrated in the bursa of Fabricius (BF), spleen, thymus, bone marrow, liver, kidney, lung and heart, indicating that infection can be multisystemic. In some birds, virus DNA was present in very large quantities, most notably in the BF, liver and small intestine. With the exception of BF and thymus, there were no histological findings that would have suggested the presence of such quantities of circovirus DNA. In view of the very large quantities of virus DNA labelling present in some tissues, and by analogy to porcine circovirus type 2 infection and psittacine beak and feather virus infections, which are known to cause severe disease, and which have similar virus distribution to that found in our geese, it seems probable that the circovirus was important in the disease manifestations shown by the infected geese. PMID:16191706

  4. Identification of canine glial cells by nonradioactive in situ hybridization.

    PubMed

    Graber, H U; Zurbriggen, A; Vandevelde, M

    1993-01-01

    Studies on the development of the canine central nervous system and on demyelinating diseases demand unequivocal identification of the glial cells. For that reason, nonradioactive in situ hybridization (ISH) was performed in primary dog brain cell cultures (DBCC) and in brain sections of neonatal dogs. Specific RNA probes were used to detect messenger RNA (mRNA) coding for proteolipid protein (PLP), myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). PLP and MBP are markers for oligodendrocytes, GFAP for astrocytes. Oligodendrocytes positive for PLP and MBP mRNA were found in both DBCC and brain sections of neonatal dogs. Astrocytes expressing GFAP specific mRNA were detected in DBCC and in brain sections. These cells were evenly distributed in the white matter with additional accumulation in the membrana limitans gliae superficialis, around the ventricles and blood vessels. ISH clearly improves the study of oligodendrocytes in brain sections as, in contrast to the immunohistochemical methods, this technique allows to identify individual cells. PMID:8135072

  5. Pallister-Killian syndrome detected by fluorescence in situ hybridization

    SciTech Connect

    Butler, M.G.; Dev, V.G.

    1995-07-03

    The Pallister-Killian syndrome is a rare cytogenetic condition first described in 1977 by Pallister et al. in 3 adults; the first affected child was reported in 1981. This syndrome (also known as Pallister mosaic aneuploidy syndrome or isochromosome 12p mosaicism) is characterized by postnatal growth retardation, seizures, hypotonia, deafness, profound mental retardation, minimal speech development, and a distinctive facial appearance (high prominent forehead, ocular hypertelorism, sparse anterior scalp hair, prominent lower lip, large ears with thick protruding lobules, cupid-bow shaped upper lip, and a long philtrum). A chromosome 12 abnormality (tetrasomy 12p) has been reported in skin biopsies from these patients but this chromosome anomaly is usually not found (or in only a small proportion, e.g., <0.5%, of blood cells) in peripheral blood. We report on an additional patient with Pallister-Killian syndrome confirmed with fluorescence in situ hybridization (FISH) using an alpha satellite DNA probe for chromosome 12. This report further illustrates the application of FISH in identifying the source of chromosomal markers of unknown origin in infants with multiple congenital anomalies specifically before the natural history of a condition allows for definitive diagnosis based on clinical findings. 9 refs., 2 figs.

  6. The Application of Fluorescence In Situ Hybridization in Different Ploidy Levels Cross-Breeding of Lily

    PubMed Central

    Wang, Qing; Wang, Jingmao; Zhang, Yiying; Zhang, Yue; Xu, Shunchao; Lu, Yingmin

    2015-01-01

    21 crossing were conducted between Asiatic Lily with different ploidy levels, the results showed that the interploidy hybridization between diploid and tetraploid lilies was not as successful as intraploidy hybridization. Regardless of male sterility, triploid lilies could be used as female parents in the hybridization which the progenies were aneuploidy. 3x×4x crosses could be cultured more successfully than 3x×2x crosses. 45S rDNA was mapped on the chromosomes of seven Lilium species and their progenies using fluorescence in situ hybridization (FISH). FISH revealed six to sixteen 45S rDNA gene loci, and normally the sites were not in pairs. The asymmetry indexes of LA (Longiflorum hybrids × Asiatic hybrids) hybrids was higher than Asiatic hybrids, the evolution degree was LA hybrids > Asiatic hybrids. 45S rDNA distributed variably on chromosome 1-10 and 12 among Asiatic hybrids. Chromosome 1 had invariable sites of 45S rDNA in all Asiatic hybrids, which could be considered as the characteristic of Asiatic hybrids. LA hybrid ‘Freya’ had two sites of 45S rDNA on one homologous chromosome 5, and also it could be found in the progenies. The karyotype and fluorescence in situ hybridization with 45S rDNA as probe were applied to identify the different genotypes of 9 hybrids. Typical chromosomes with parental signal sites could be observed in all the genotypes of hybrids, it was confirmed that all the hybrids were true. PMID:26010356

  7. Determination of HER2 amplification in primary breast cancer using dual-colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens

    PubMed Central

    García-Caballero, Tomás; Grabau, Dorthe; Green, Andrew R; Gregory, John; Schad, Arno; Kohlwes, Elke; Ellis, Ian O; Watts, Sarah; Mollerup, Jens

    2010-01-01

    García-Caballero T, Grabau D, Green A R, Gregory J, Schad A, Kohlwes E, Ellis I O, Watts S & Mollerup J (2010) Histopathology56, 472–480 Determination of HER2 amplification in primary breast cancer using dual-colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens Aims: Fluorescence in situ hybridization (FISH) can be used to reveal several genomic imbalances relevant to proper cancer diagnosis and to the correct treatment regime. However, FISH requires expensive and advanced fluorescence microscopes in addition to expertise in fluorescence microscopy. To determine whether a newly developed dual-colour chromogenic in situ hybridization (CISH) method is a suitable alternative to FISH, we analysed the human epidermal growth factor receptor 2 gene (HER2) amplification level of 168 breast cancer specimens using dual-colour CISH and FISH and compared the results. Methods and results: We found 100% agreement between HER2 status determined by FISH and dual-colour CISH. Furthermore, we observed that the time used to score slides was significantly reduced by 28% in dual-colour CISH compared with the FISH protocol. Concordance between HER2 protein status and dual-colour CISH or FISH was equally good with an overall agreement of 96.8%. Correlation between the HER2/centromere 17 gene ratios obtained with dual-colour CISH and FISH was highly significant with an overall correlation coefficient (ρ) of 0.96. Conclusions: We conclude that dual-colour CISH and bright field microscopy are excellent alternatives to FISH when analysing the HER2 status of primary breast cancer. PMID:20459554

  8. Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

    PubMed Central

    Chen, Chun-Chun; Wada, Kazuhiro; Jarvis, Erich D.

    2012-01-01

    Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the

  9. Detection of dengue group viruses by fluorescence in situ hybridization

    PubMed Central

    2012-01-01

    Background Dengue fever (DF) and dengue hemorrhagic fever (DHF) represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus) that comprises four distinct serotypes (DENV-1 to DENV-4). Fluorescence in situ hybridization (FISH) has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays. Methods Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae). The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy. Results The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes. Conclusion Based on the FISH technique, we propose a way to design and use oligonucleotide probes to

  10. Role of fluorescence in situ hybridization (FISH) in sequencing the tomato genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chromosomes at various stages of the cell cycle can be used for localization of DNA probes via Fluorescence in situ hybridization (FISH). While mitotic metaphase chromosomes are demonstrably too short and compact for this purpose, long pachytene chromosomes are ideal. BACs that hybridize to euchrom...

  11. Stillwater Hybrid Geo-Solar Power Plant Optimization Analyses

    SciTech Connect

    Wendt, Daniel S.; Mines, Gregory L.; Turchi, Craig S.; Zhu, Guangdong; Cohan, Sander; Angelini, Lorenzo; Bizzarri, Fabrizio; Consoli, Daniele; De Marzo, Alessio

    2015-09-02

    The Stillwater Power Plant is the first hybrid plant in the world able to bring together a medium-enthalpy geothermal unit with solar thermal and solar photovoltaic systems. Solar field and power plant models have been developed to predict the performance of the Stillwater geothermal / solar-thermal hybrid power plant. The models have been validated using operational data from the Stillwater plant. A preliminary effort to optimize performance of the Stillwater hybrid plant using optical characterization of the solar field has been completed. The Stillwater solar field optical characterization involved measurement of mirror reflectance, mirror slope error, and receiver position error. The measurements indicate that the solar field may generate 9% less energy than the design value if an appropriate tracking offset is not employed. A perfect tracking offset algorithm may be able to boost the solar field performance by about 15%. The validated Stillwater hybrid plant models were used to evaluate hybrid plant operating strategies including turbine IGV position optimization, ACC fan speed and turbine IGV position optimization, turbine inlet entropy control using optimization of multiple process variables, and mixed working fluid substitution. The hybrid plant models predict that each of these operating strategies could increase net power generation relative to the baseline Stillwater hybrid plant operations.

  12. Denaturation, renaturation, and loss of DNA during in situ hybridization procedures.

    PubMed

    Raap, A K; Marijnen, J G; Vrolijk, J; van der Ploeg, M

    1986-05-01

    With the aim of optimizing in situ hybridization methods, alkaline, acid, and thermal denaturation procedures have been studied for their ability to separate the DNA strands of nuclear DNA and for the DNA losses they induce. Isolated methanol/acetic acid-fixed mouse liver nuclei have been used as a biological object. The results, obtained with acridine orange staining and microfluorometry, show that all denaturations studied lead to almost complete strand separation. Quantitative DNA staining and cytometry indicated that with heat and alkaline denaturation about 40% of the DNA is lost. Acid denaturation led to about 20% DNA loss. For the alkaline denaturation, the DNA retention could be improved to a 20% DNA loss by adding 70% ethanol to the denaturation medium. During hybridization, another 20% DNA loss occurs. When denatured nuclei are brought under annealing conditions, a rapid renaturation of a considerable fraction of the remaining DNA occurs. The extent of renaturation was dependent on the type of denaturation used. For the ethanolic alkaline denaturation, it was estimated to be 35%. Quantitative nonautoradiographic in situ hybridization experiments with acetylaminofluorene-modified mouse satellite DNA showed that alkaline denaturation procedures are superior to the heat and acid denaturation. As proven by acridine orange fluorescence measurements, hybridization conditions can be designed that permit DNA.RNA hybridization under in situ DNA.DNA denaturing conditions. These conditions should be very useful, especially for in situ hybridization with single-stranded RNA probes. PMID:3709305

  13. Fluorescence in situ hybridization analysis is a helpful test for the diagnosis of dermatofibrosarcoma protuberans.

    PubMed

    Karanian, Marie; Pérot, Gaëlle; Coindre, Jean-Michel; Chibon, Frédéric; Pedeutour, Florence; Neuville, Agnès

    2015-02-01

    Cytogenetically, most dermatofibrosarcoma protuberans are characterized by chromosomal rearrangements resulting in the collagen type-1 alpha 1 (COL1A1)-platelet-derived growth factor β (PDGFB) fusion gene. This abnormality can be detected by fluorescence in situ hybridization (FISH) analysis in routine practice. The aim of this study was to evaluate the role of the FISH analysis in the diagnosis of dermatofibrosarcoma protuberans. A FISH analysis was prospectively and systematically performed on a series of 448 consecutive tumor specimens. All cases were reviewed by two independent pathologists and classified in three categories according to the probability of a DFSP diagnosis before molecular analyses. Cases were classified as certain when dermatofibrosarcoma protuberans was the only possible diagnosis. Those cases for which dermatofibrosarcoma protuberans remained the first diagnosis, but other differential diagnosis existed, were regarded as probable. When dermatofibrosarcoma protuberans was considered a differential diagnosis, they were labeled as possible. The final diagnosis was supported by clinicopathological findings and results of FISH analyses. Immunohistochemical analysis of CD34 was systematically performed, and additional markers when necessary. The cases (n=37) with a non-interpretable FISH were excluded. For the 185 certain tumors specimens: 178 (96%) FISH analyses showed a PDGFB/COL1A1 rearrangement, 7 (4%) were negative. For the 114 probable tumors specimens: 104 (91%) FISH analyses were positive and 10 (9%) were negative leading to a new diagnosis in 8 cases. For the 112 possible cases: 91 (81%) FISH analyses were negative and 21 (19%) were positive. Of the 21 cases, initial diagnoses included unclassified sarcoma, myxofibrosarcoma, dermatofibroma, reactive lesion, solitary fibrous tumor, perineurioma, benign nerve sheath tumor, and undifferentiated spindle cell tumor without malignant evidence. FISH analysis has been helpful for confirming the

  14. [Non-radioactive in situ hybridization of alpha-satellite sequences in cytogenetic diagnosis].

    PubMed

    Perfumo, C; Arslanian, A; Zara, F; Piombo, G; Pierluigi, M

    1992-01-01

    Non isotopic in situ hybridization with alpha-satellite DNA probes in the cytogenetic diagnosis. Standard banding cytogenetic techniques do not always allow to define the structure and the origin of chromosome rearrangements involving the centromere region. Non-isotopic in situ hybridization of alphoid sequences has allowed to determine the origin of the centromeres in the metaphases of 5 patients referred to us for: 2 structural rearrangements involving chromosome 21, 2 structural rearrangements involving chromosome Y and 1 reciprocal translocation involving on chromosome 20 and one chromosome 15. PMID:1465321

  15. Distribution and Evolution of Repeated Sequences in Genomes of Triatominae (Hemiptera-Reduviidae) Inferred from Genomic In Situ Hybridization

    PubMed Central

    Pita, Sebastian; Panzera, Francisco; Sánchez, Antonio; Panzera, Yanina; Palomeque, Teresa; Lorite, Pedro

    2014-01-01

    The subfamily Triatominae, vectors of Chagas disease, comprises 140 species characterized by a highly homogeneous chromosome number. We analyzed the chromosomal distribution and evolution of repeated sequences in Triatominae genomes by Genomic in situ Hybridization using Triatoma delpontei and Triatoma infestans genomic DNAs as probes. Hybridizations were performed on their own chromosomes and on nine species included in six genera from the two main tribes: Triatomini and Rhodniini. Genomic probes clearly generate two different hybridization patterns, dispersed or accumulated in specific regions or chromosomes. The three used probes generate the same hybridization pattern in each species. However, these patterns are species-specific. In closely related species, the probes strongly hybridized in the autosomal heterochromatic regions, resembling C-banding and DAPI patterns. However, in more distant species these co-localizations are not observed. The heterochromatic Y chromosome is constituted by highly repeated sequences, which is conserved among 10 species of Triatomini tribe suggesting be an ancestral character for this group. However, the Y chromosome in Rhodniini tribe is markedly different, supporting the early evolutionary dichotomy between both tribes. In some species, sex chromosomes and autosomes shared repeated sequences, suggesting meiotic chromatin exchanges among these heterologous chromosomes. Our GISH analyses enabled us to acquire not only reliable information about autosomal repeated sequences distribution but also an insight into sex chromosome evolution in Triatominae. Furthermore, the differentiation obtained by GISH might be a valuable marker to establish phylogenetic relationships and to test the controversial origin of the Triatominae subfamily. PMID:25478792

  16. Fluorescence in situ hybridization and spectral imaging of coral-associated bacterial communities.

    PubMed

    Ainsworth, T D; Fine, M; Blackall, L L; Hoegh-Guldberg, O

    2006-04-01

    Microbial communities play important roles in the functioning of coral reef communities. However, extensive autofluorescence of coral tissues and endosymbionts limits the application of standard fluorescence in situ hybridization (FISH) techniques for the identification of the coral-associated bacterial communities. This study overcomes these limitations by combining FISH and spectral imaging. PMID:16598010

  17. Detection of resistance to macrolides in thermotolerant campylobacter species by fluorescence in situ hybridization.

    PubMed

    Haas, Michaela; Essig, Andreas; Bartelt, Edda; Poppert, Sven

    2008-11-01

    The resistance of enteritis-causing Campylobacter strains to erythromycin is an emerging problem. We therefore evaluated fluorescence in situ hybridization (FISH) for the rapid detection of resistance using 74 campylobacter isolates. FISH showed specificity and sensitivity of 100% for the detection of high-level resistance. PMID:18753354

  18. Specific Detection and Localization of Microsporidian Parasites in Invertebrate Hosts by Using In Situ Hybridization

    PubMed Central

    Smith, Judith E.; Solter, Leellen; Perotti, M. Alejandra; Braig, Henk R.; Dunn, Alison M.

    2013-01-01

    We designed fluorescence in situ hybridization probes for two distinct microsporidian clades and demonstrated their application in detecting, respectively, Nosema/Vairimorpha and Dictyoceola species. We used them to study the vertical transmission of two microsporidia infecting the amphipod Gammarus duebeni. PMID:23087031

  19. Quantitative in situ hybridization for the study of gene expression at the regional and cellular levels.

    PubMed

    Le Moine, Catherine

    2003-08-01

    Quantitative in situ hybridization allows measurement of mRNA level modifications in a variety of experimental conditions. This analysis may be performed both at the regional anatomical and cellular levels by densitometry, neuronal counting and silver grain measurements. PMID:18428577

  20. In situ hybridization and immunohistochemistry for the detection of porcine cytomegalovirus.

    PubMed

    Sekiguchi, Maki; Shibahara, Tomoyuki; Miyazaki, Ayako; Tajima, Tomoko; Shimizu, Shinya; Kabali, Emmanuel; Takano, Yoshiyuki; Sasaki, Yosuke; Kubo, Masanori

    2012-01-01

    To establish in situ hybridization and immunohistochemistry based-assays for the detection of porcine cytomegalovirus, routinely processed renal tissue sections from 34 diseased piglets suspected of having the infection were obtained and examined. Using hematoxylin and eosin, porcine cytomegalovirus inclusion bodies were found in the nucleus of renal epithelial cells and capillary endothelial cells in the renal medulla in 30 cases. Inclusion bodies corresponding to porcine cytomegalovirus mRNA after in situ hybridization or porcine cytomegalovirus antigens after immunohistochemistry were easily determined. The cells were characterized by cytomegaly and basophilic intranuclear inclusion bodies. Using in situ hybridization, porcine cytomegalovirus mRNA were clearly detected in the nucleus and cytoplasm of the cells in 28 of the 30 (93.3%) cases. Using immunohistochemistry, porcine cytomegalovirus antigens were clearly detected in the cytoplasm of the cells in 21 of the 30 (70.0%) cases. Higher specificities and increased intensity of staining was observed with minimal background using in situ hybridization and immunohistochemistry compared with hematoxylin and eosin. Thus, the two established methods are useful and helpful tools for detecting the presence of a porcine cytomegalovirus infection. PMID:22008295

  1. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 Section 866.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES...

  2. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 Section 866.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES...

  3. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 Section 866.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES...

  4. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 Section 866.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES...

  5. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 Section 866.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES...

  6. Evolution of Chromosome 6 of Solanum Species Revealed by Comparative Fluorescence in Situ Hybridization Mapping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative genome mapping is an important tool in evolutionary research. Here we demonstrate a comparative fluorescent in situ hybridization (FISH) mapping strategy. A set of 13 bacterial artificial chromosome (BAC) clones derived from potato chromosome 6 was used for FISH mapping in seven differen...

  7. De Novo nonreciprocal translocation 1;8 confirmed by fluorescent in situ hybridization

    SciTech Connect

    Wiley, J.E.; Stout, C.; Palmer, S.M.

    1995-07-17

    Constitutional nonreciprocal translocations are extremely rare, and even their existence is controversial. We report on a newborn infant with a de novo nonreciprocal translocation between chromosomes 1 and 8 resulting in 1q42.3 deletion syndrome. Fluorescent in situ hybridization with whole chromosome paints confirmed the conventional cytogenetic diagnosis. 3 refs., 2 figs., 1 tab.

  8. [Use of photo-anchoring of DNA probes for fluorescent in situ hybridization].

    PubMed

    Nasedkina, T V; Mal'kov, R B; Fedorova, L I; Godovikova, T S; Kolpashchikov, D M; Poletaev, A I

    1998-01-01

    A possibility was investigated to use photo-crosslinking DNA probes for fluorescent in situ hybridization (FISH). DNA probes were modified by incorporating photonucleotides in these, containing a photoreactive group (tetrafluorobenzazid) and capable of making covalent bonds with the examined DNA, when irradiated in 300-330 nm region. The photonucleotide was incorporated into the probe either by nick-translation, or upon elongation of the hybridized probe by the Kljonow fragment. It has been shown that the DNA probe, cross-linking to a chromosome as a result of covalent bonds, is not removed from the place of hybridization under consequent denaturating washing, which makes it possible to carry out the following DNA hybridization with selective conservation of signals obtained due to previous hybridization. This peculiarity of photo-linking DNA probes makes it possible to use them for the two-step DNA hybridization. To demonstrate this, preparations of human chromosomes were investigated. On the first step, chromosomal DNA was hybridized by means of DNA probe having nucleotide sequences of centromeric regions of chromosomes 13 and 21, the probe being linked to chromosomal DNA by the photonucleotide. Following the denaturation treatment of the preparation, and after the second chromosomal DNA hybridization with cosmid DNA, containing chromosome 13 DNA nucleotide sequence, the signal in chromosome 13 centromeric region was retained to serve a marker of this chromosome, thus fascilitating its easier identification following the hybridization of its DNA with cosmic DNA. The denaturation stability of photo-crosslinking probes opens some new possibilities in technology of DNA in situ hybridization. PMID:9821246

  9. Demonstration of tissue-specific gene expression by in situ hybridization

    SciTech Connect

    Angerer, L.M.; Cox, K.H.; Angerer, R.C.

    1987-01-01

    In situ hybridization has emerged as a valuable tool for the identification of individual cells expressing specific genes. Recently, methods have become sufficiently sensitive to detect mRNAs present at the level of only a few molecules per cell. When mRNAs are expressed in only a small fraction of the cells in a mixed population, in situ hybridization may be the most sensitive nucleic acid hybridization technique available. The authors' laboratory has shown that antisense RNA probes offer a unique combination of advantages for detection of individual mRNAs by in situ hybridization. Most importantly, antisense probes provide a large increase in sensitivity due to the absence of competing probe self-reassociation. The high stability of RNA-RNA duplexes allows use of higher post hybridization wash temperatures to achieve a given fidelity of base pairing (stringency), which also reduced backgrounds. RNA transcripts can be synthesized from truncated templates such that they are essentially devoid of vector sequences. This sequence purity maximizes the signal to noise ratio since lower probe concentrations are required to saturate target RNAs.

  10. In situ hybridization of phytoplankton using fluorescently labeled rRNA probes.

    PubMed

    Groben, René; Medlin, Linda

    2005-01-01

    Phytoplankton are one of the major components of ecosystem processes and play an important role in many biogeochemical cycles in the marine and freshwater environment. Despite their importance, many microalgae are poorly described and little is known of broad spatial and temporal scale trends in their abundance and distribution. Reasons for this are that microalgae are often small, lack distinct morphological features, and are unculturable, which make analyses difficult. It is now possible by using molecular biological techniques to advance our knowledge of aquatic biodiversity and to understand how biodiversity supports ecosystem structure, dynamics, and resilience. We present in this chapter a brief review of the progress that has been made in analyzing microalgae from populations to the species level. The described methods range from DNA fingerprinting techniques, such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs), to microsatellites, which are used in population studies, to sequence analysis, which help to reconstruct the evolutionary history of organisms and to examine relationships at various taxonomic levels. Special emphasis is given to the application of molecular probes for the identification and characterization of microalgal taxa. The fast and secure identification of phytoplankton, especially of toxic species, is important from an ecological and economical point of view and whole-cell hybridization with specific fluorochrome-labeled probes followed by fluorescence microscopy or flow cytometry offers a fast method for this purpose. In this context, we present a detailed protocol for fluorescence in situ hybridization (FISH) of ribosomal RNA (rRNA) probes that can be applied to many algal cell types and discuss practical considerations of its use. PMID:15865974

  11. Prediction of hybrid performance in maize using molecular markers and joint analyses of hybrids and parental inbreds.

    PubMed

    Schrag, Tobias A; Möhring, Jens; Melchinger, Albrecht E; Kusterer, Barbara; Dhillon, Baldev S; Piepho, Hans-Peter; Frisch, Matthias

    2010-01-01

    The identification of superior hybrids is important for the success of a hybrid breeding program. However, field evaluation of all possible crosses among inbred lines requires extremely large resources. Therefore, efforts have been made to predict hybrid performance (HP) by using field data of related genotypes and molecular markers. In the present study, the main objective was to assess the usefulness of pedigree information in combination with the covariance between general combining ability (GCA) and per se performance of parental lines for HP prediction. In addition, we compared the prediction efficiency of AFLP and SSR marker data, estimated marker effects separately for reciprocal allelic configurations (among heterotic groups) of heterozygous marker loci in hybrids, and imputed missing AFLP marker data for marker-based HP prediction. Unbalanced field data of 400 maize dent x flint hybrids from 9 factorials and of 79 inbred parents were subjected to joint analyses with mixed linear models. The inbreds were genotyped with 910 AFLP and 256 SSR markers. Efficiency of prediction (R (2)) was estimated by cross-validation for hybrids having no or one parent evaluated in testcrosses. Best linear unbiased prediction of GCA and specific combining ability resulted in the highest efficiencies for HP prediction for both traits (R (2) = 0.6-0.9), if pedigree and line per se data were used. However, without such data, HP for grain yield was more efficiently predicted using molecular markers. The additional modifications of the marker-based approaches had no clear effect. Our study showed the high potential of joint analyses of hybrids and parental inbred lines for the prediction of performance of untested hybrids. PMID:19916002

  12. Dissolved methane profiles in marine sediments observed in situ differ greatly from analyses of recovered cores

    NASA Astrophysics Data System (ADS)

    Zhang, X.; Brewer, P. G.; Hester, K.; Ussler, W.; Walz, P. M.; Peltzer, E. T.; Ripmeester, J.

    2009-12-01

    The flux of dissolved methane through continental margin sediments is of importance in marine geochemistry due to its role in massive hydrate formation with enigmatic climate consequences, and for the huge and complex microbial assemblage it supports. Yet the actual dissolved methane concentration driving this flux is poorly known since strong degassing during sample recovery from depth is commonplace. Thus, pore water analyses from high CH4 environments typically show values clustered around the one-atmosphere equilibrium value of 1-2 mM, erasing the original pore water profile and frustrating model calculations. We show that accurate measurement of pore water profiles of dissolved CH4, SO4, and H2S can be made rapidly in situ using a Raman-based probe. While Raman spectra were formerly believed to yield only qualitative data we show that by using a peak area ratio technique to known H2O bands and a form of Beer’s Law quantitative data may be readily obtained. Results from Hydrate Ridge, Oregon clearly show coherent profiles of all three species in this high flux environment, and while in situ Raman and conventional analyses of SO4 in recovered cores agree well, very large differences in CH4 are found. The in situ CH4 results show up to 35 mM in the upper 30cm of seafloor sediments and are inversely correlated with SO4. This is below the methane hydrate saturation value, yet disturbing the sediments clearly released hydrate fragments suggesting that true saturation values may exist only in the hydrate molecular boundary layer, and that lower values may typically characterize the bulk pore fluid of hydrate-hosting sediments. The in situ Raman measurement protocols developed take only a few minutes. Profiles obtained in situ showed minimal fluorescence while pore water samples from recovered cores quickly developed strong fluorescence making laboratory analyses using Raman spectroscopy challenging and raising questions over the reaction sequence responsible for

  13. The value of immunohistochemistry and in situ hybridization in detecting cytomegalovirus in bone marrow transplant recipients.

    PubMed

    Rasing, L A; De Weger, R A; Verdonck, L F; van der Bij, W; Compier-Spies, P I; De Gast, G C; Van Basten, C D; Schuurman, H J

    1990-06-01

    Autopsy tissues of 19 patients with complications after bone marrow transplantation (BMT) were analysed for the presence of cytomegalovirus (CMV) using histochemical methods. CMV antigens were detected by antibodies to CMV Immediate Early Antigen (IEA) or CMV Late Antigen (LA). CMV-DNA was detected by DNA in situ hybridization (DISH). IEA was detected in one or more tissues in 79% of 14 patients from whom frozen tissue was available. CMV-DNA was detected on paraffin sections in 84% of all 19 patients. CMV components were present in all organs studied; the highest incidence was found in lung, gastrointestinal tract and kidney. In histology, only 37% of patients showed signs of CMV infection by the presence of cytomegalic cells with nuclear inclusions (or so called "owl eye cells"). In tissue culture, only 33% of 15 patients were CMV positive. Serologically, 68% of all patients had active CMV infection, as indicated by a rise in antibody titres. We conclude that the quick detection of CMV IEA and CMV-DNA has a high sensitivity and predictive value, which is comparable to or exceeds the serological detection of CMV. PMID:2166539

  14. Phylogenetic analysis of multiprobe fluorescence in situ hybridization data from tumor cell populations

    PubMed Central

    Schwartz, Russell

    2013-01-01

    Motivation: Development and progression of solid tumors can be attributed to a process of mutations, which typically includes changes in the number of copies of genes or genomic regions. Although comparisons of cells within single tumors show extensive heterogeneity, recurring features of their evolutionary process may be discerned by comparing multiple regions or cells of a tumor. A useful source of data for studying likely progression of individual tumors is fluorescence in situ hybridization (FISH), which allows one to count copy numbers of several genes in hundreds of single cells. Novel algorithms for interpreting such data phylogenetically are needed, however, to reconstruct likely evolutionary trajectories from states of single cells and facilitate analysis of tumor evolution. Results: In this article, we develop phylogenetic methods to infer likely models of tumor progression using FISH copy number data and apply them to a study of FISH data from two cancer types. Statistical analyses of topological characteristics of the tree-based model provide insights into likely tumor progression pathways consistent with the prior literature. Furthermore, tree statistics from the resulting phylogenies can be used as features for prediction methods. This results in improved accuracy, relative to unstructured gene copy number data, at predicting tumor state and future metastasis. Availability: Source code for software that does FISH tree building (FISHtrees) and the data on cervical and breast cancer examined here are available at ftp://ftp.ncbi.nlm.nih.gov/pub/FISHtrees. Contact: sachowdh@andrew.cmu.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23812984

  15. Nanogold In Situ Hybridization for Phylogenetic Identification in Geologic Samples Using Environmental Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Ehrhardt, C.; Haymon, R.; Sievert, S.; Holden, P.

    2006-12-01

    Collecting phylogenetic information simultaneously with mineral textures and associations for geomicrobiological studies has always been a challenge. Recently a new type of nucleotide reporter system has been developed that utilizes small particles of nanogold (1.4 nm) covalently attached to oligonucelotide probes. Due to the small size and electron density of these nanogold reporter molecules, this in situ hybridization technique allows for the phylogenetic identification of microbial targets with a scanning electron microscope. Here we present new applications of the nanogold hybridization technique for pure cultures and natural microbial communities in a range of geologic samples including sand grains, basalt chips incubated on deep sea hydrothermal vents, and gypsum crusts sampled from a saline lake. While we do observe nonspecific binding of nanogold probes to minerals and organic compounds in geologic matrices, this can be distinguished from positive hybridization events with a spatial variety analysis. To assess the potential of nanogold hybridizations for quantitative assessments of microbial communities, fluorescent in situ hybridizations (FISH) were performed on all samples and compared to cell counts generated from nanogold hybridizations.

  16. Coordinated In Situ Nanosims Analyses of H-C-O Isotopes in ALH 84001 Carbonates

    NASA Technical Reports Server (NTRS)

    Usui, T.; Alexander, C. M. O'D.; Wang, J.; Simon, J. I.; Jones, J. H.

    2016-01-01

    The surface geology and geomorphology of Mars indicate that it was once warm enough to maintain a large body of liquid water on its surface, though such a warm environment might have been transient. This study reports the hydrogen, carbon, and oxygen isotope compositions of the ancient atmosphere/hydrosphere of Mars based on in situ ion microprobe analyses of approximately 4 Ga-old carbonates in Allan Hills (ALH) 84001. The ALH 84001 carbonates are the most promising targets because they are thought to have formed from fluid that was closely associated with the Noachian atmosphere. While there are a number of carbon and oxygen isotope studies of the ALH 84001 carbonates, in situ hydrogen isotope analyses of these carbonates are limited and were reported more than a decade ago. Well-documented coordinated in situ analyses of carbon, oxygen and hydrogen isotopes provide an internally consistent dataset that can be used to constrain the nature of the Noachian atmosphere/hydrosphere and may eventually shed light on the hypothesis of ancient watery Mars.

  17. Evolution in situ: hybrid origin and establishment of willows (Salix L.) on alpine glacier forefields

    PubMed Central

    Gramlich, S; Sagmeister, P; Dullinger, S; Hadacek, F; Hörandl, E

    2016-01-01

    Little attention has been paid to the evolutionary consequences of the colonizing dynamics and succession processes following glacier retreat. Here we studied hybrid populations that have recently formed and established on glacier forefields of the European Alps owing to secondary contact of a lowland colonizer with a subalpine species. We analyzed the composition of two hybrid populations between Salix purpurea and Salix helvetica with nine microsatellite markers by using Bayesian methods (structure and NewHybrids), and simulations. We also studied niche differentiation between the hybrids and the parental species based on indicator values, soil pH and water retention potential measurements. Allelic structure of hybrids confirms the assumed parentage and in situ origin of the crosses on two independent sites within the last decades. Both hybrid populations comprised F1 and later generation hybrids (F2 and backcrosses), confirming hybrid fertility. The parental species showed significant differences in niche characteristics for temperature, soil pH, nutrients and moisture. Remarkably, the hybrids exhibited a higher tolerance to cold temperatures, nutrient-poor and acidic soils than either parent. Our results show that willow hybrids originated after glacier retreat and have established persistent populations within a few decades. One factor contributing to hybrid establishment in sympatry with their parents is their ability to occupy more extreme niches than either parental species within a mosaic-like pattern of microhabitats on the forefield. Introgression and/or transgressive segregation may have resulted in novel genotypes that are able to expand the ecological spectrum of either parent. PMID:26980342

  18. Evolution in situ: hybrid origin and establishment of willows (Salix L.) on alpine glacier forefields.

    PubMed

    Gramlich, S; Sagmeister, P; Dullinger, S; Hadacek, F; Hörandl, E

    2016-06-01

    Little attention has been paid to the evolutionary consequences of the colonizing dynamics and succession processes following glacier retreat. Here we studied hybrid populations that have recently formed and established on glacier forefields of the European Alps owing to secondary contact of a lowland colonizer with a subalpine species. We analyzed the composition of two hybrid populations between Salix purpurea and Salix helvetica with nine microsatellite markers by using Bayesian methods (structure and NewHybrids), and simulations. We also studied niche differentiation between the hybrids and the parental species based on indicator values, soil pH and water retention potential measurements. Allelic structure of hybrids confirms the assumed parentage and in situ origin of the crosses on two independent sites within the last decades. Both hybrid populations comprised F1 and later generation hybrids (F2 and backcrosses), confirming hybrid fertility. The parental species showed significant differences in niche characteristics for temperature, soil pH, nutrients and moisture. Remarkably, the hybrids exhibited a higher tolerance to cold temperatures, nutrient-poor and acidic soils than either parent. Our results show that willow hybrids originated after glacier retreat and have established persistent populations within a few decades. One factor contributing to hybrid establishment in sympatry with their parents is their ability to occupy more extreme niches than either parental species within a mosaic-like pattern of microhabitats on the forefield. Introgression and/or transgressive segregation may have resulted in novel genotypes that are able to expand the ecological spectrum of either parent. PMID:26980342

  19. Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: Clinical experience with 4,500 specimens

    SciTech Connect

    Ward, B.E.; Gersen, S.L.; Carelli, M.P.; McGuire, N.M.; Dackowski, W.R.; Klinger, K.W. ); Weinstein, M. ); Sandlin, C. ); Klinger, K.W. )

    1993-05-01

    Detection of chromosome aneuploidies in uncultured amniocytes is possible using fluorescence in situ hybridization (FISH). The authors herein describe the results of the first clinical program which utilized FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes. FISH was performed on physician request, as an adjunct to cytogenetics in 4,500 patients. Region-specific DNA probes to chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. A sample was considered to be euploid when all autosomal probes generated two hybridization signals and when a normal sex chromosome pattern was observed in greater than or equal to 80% of hybridized nuclei. A sample was considered to be aneuploid when greater than or equal to 70% of hybridized nuclei displayed the same abnormal hybridization pattern for a specific probe. Of the attempted analyses, 90.2% met these criteria and were reported as informative to referring physicians within 2 d of receipt. Based on these reporting parameters, the overall detection rate for aneuploidies was 73.3% (107/146), with an accuracy of informative results for aneuploidies of 93.9% (107/114). Compared to cytogenetics, the accuracy of all informative FISH results, euploid and aneuploid, was 99.8%, and the specificity was 99.9%. In those pregnancies where fetal abnormalities had been observed by ultrasound, referring physicians requested FISH plus cytogenetics at a significantly higher rate than they requested cytogenetics alone. The current prenatal FISH protocol is not designed to detect all chromosome abnormalities and should only be utilized as an adjunctive test to cytogenetics. This experience demonstrates that FISH can provide a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies. 40 refs., 1 fig., 5 tabs.,

  20. Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: clinical experience with 4,500 specimens.

    PubMed Central

    Ward, B E; Gersen, S L; Carelli, M P; McGuire, N M; Dackowski, W R; Weinstein, M; Sandlin, C; Warren, R; Klinger, K W

    1993-01-01

    Detection of chromosome aneuploidies in uncultured amniocytes is possible using fluorescence in situ hybridization (FISH). We herein describe the results of the first clinical program which utilized FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes. FISH was performed on physician request, as an adjunct to cytogenetics in 4,500 patients. Region-specific DNA probes to chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. A sample was considered to be euploid when all autosomal probes generated two hybridization signals and when a normal sex chromosome pattern was observed in greater than or equal to 80% of hybridized nuclei. A sample was considered to be aneuploid when greater than or equal to 70% of hybridized nuclei displayed the same abnormal hybridization pattern for a specific probe. Of the attempted analyses, 90.2% met these criteria and were reported as informative to referring physicians within 2 d of receipt. Based on these reporting parameters, the overall detection rate for aneuploidies was 73.3% (107/146), with an accuracy of informative results for aneuploidies of 93.9% (107/114). Compared to cytogenetics, the accuracy of all informative FISH results, euploid and aneuploid, was 99.8%, and the specificity was 99.9%. In those pregnancies where fetal abnormalities had been observed by ultrasound, referring physicians requested FISH plus cytogenetics at a significantly higher rate than they requested cytogenetics alone. The current prenatal FISH protocol is not designed to detect all chromosome abnormalities and should only be utilized as an adjunctive test to cytogenetics. This experience demonstrates that FISH can provide a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies. PMID:8488836

  1. [Three cases of vulvar bowenoid papulosis: the localization of HPV DNA by in situ hybridization].

    PubMed

    Kioka, H; Nagai, N; Tanioka, Y; Fujii, T; Katsube, Y; Egawa, K; Fujiwara, A

    1989-09-01

    Cytological, histological, and molecular biological studies were conducted in 3 cases of vulvar Bowenoid papulosis, using biotinylated HPV DNA probes by in situ hybridization. 1) Cytological findings showed dyskaryotic cells that revealed hyperchromatism with a coarse granular pattern, and a high N/C ratio was observed among the dyskeratotic cells. 2) In 2 cases of Bowenoid papulosis lesions, HPV 16 DNA was detected in the nucleus of the dysplastic cells. 3) In one case of Bowenoid papulosis, a complicated carcinoma in situ of the uterine cervix was observed, and the HPV 16 DNA was found to be positive in both the vulva and cervix. PMID:2550688

  2. Hybrid in situ replacement for Samson group V Staphylococcus aureus aortic graft infection.

    PubMed

    Karpenko, A A; Ignatenko, P V; Beliaev, A M

    2013-01-01

    Aortic prosthesis replacements including extra-anatomical bypass procedures, in situ revascularisations with the neoaortoiliac system, antibiotic bounded prostheses or allogeneic grafts have high graft reinfection rates. We described a case of a 68-year-old man with Samson group V Staphylococcus aureus infection of his aortobifemoral graft. He underwent an explantation of the infected graft, wound debridement and a hybrid in situ allogeneic aortoiliofemoral replacement. During surgery one of the limbs of the cryopreserved human aortic allogeneic graft was anastomosed with the endarterectomised left common iliac artery, which later was angioplastied and stented. The closed system Jackson-Pratt drains were used to prevent perigraft fluid collection. The groin wound was treated with the vacuum-assisted closure dressing. On review in 6 months he remained symptom free. We conclude that a hybrid management of infected aortic prosthesis may reduce graft reinfection. PMID:23897382

  3. Hybrid in situ replacement for Samson group V Staphylococcus aureus aortic graft infection

    PubMed Central

    Karpenko, A A; Ignatenko, P V; Beliaev, A M

    2013-01-01

    Aortic prosthesis replacements including extra-anatomical bypass procedures, in situ revascularisations with the neoaortoiliac system, antibiotic bounded prostheses or allogeneic grafts have high graft reinfection rates. We described a case of a 68-year-old man with Samson group V Staphylococcus aureus infection of his aortobifemoral graft. He underwent an explantation of the infected graft, wound debridement and a hybrid in situ allogeneic aortoiliofemoral replacement. During surgery one of the limbs of the cryopreserved human aortic allogeneic graft was anastomosed with the endarterectomised left common iliac artery, which later was angioplastied and stented. The closed system Jackson-Pratt drains were used to prevent perigraft fluid collection. The groin wound was treated with the vacuum-assisted closure dressing. On review in 6 months he remained symptom free. We conclude that a hybrid management of infected aortic prosthesis may reduce graft reinfection. PMID:23897382

  4. Assignment of the phosducin (PDC) gene to human chromosome 1q25-1q32. 1 by somatic cell hybridization and in situ hybridization

    SciTech Connect

    Sparkes, R.S.; Kojis, T.; Klisak, I.; Heinzmann, C.; Bateman, J.B. ); Lee, R.H. ); Shinohara, T. ); Craft, C.M. )

    1993-11-01

    Phosducin is a soluble photoreceptor phosphoprotein that probably modulates phototransduction in the retina and thus qualifies as a potential candidate gene for retinitis pigmentosa. Using both human/mouse somatic cell hybrids and in situ hybridization to human metaphase chromosomes, the authors have mapped this gene to chromosome 1q25-1q32.1. 18 refs., 2 figs.

  5. Combined in situ Hybridization/Immunohistochemistry (ISH/IH) on Free-floating Vibratome Tissue Sections

    PubMed Central

    Lopez, Manuel E.

    2016-01-01

    In situ hybridization and immunostaining are common techniques for localizing gene expression, the mRNA and protein respectively, within tissues. Both techniques can be applied to tissue sections to achieve similar goals, but in some cases, it is necessary to use them together. For example, complement C1q is a secreted protein complex that can target the innate immune response during inflammation. Complement has been found to be elevated early and before severe neurodegeneration in several disease models. Thus, complement may serve as an important marker for disease progression and may contribute to the pathology under certain conditions. Since complement is a secreted complex, immunostaining for C1q does not necessarily reveal where compliment is produced. In situ hybridization for complement components, C1q a, b, or c mRNA, is ideal to mark complement producing cells in tissue. In situ hybridization can be coupled with cell-type-specific immunostaining for accurate identification of the cell types involved. Protein localization and mRNA localization together can reveal details as to the relationship between complement producing and complement target cells within disease tissues. Here we outline the steps for combined in situ hybridization and immunostaining on the same tissue section. The protocol outlined here has been designed for detection of complement C1q in neurons and microglia in the mouse brain. Provided here are two approaches for combined ISH/IH. In the 1st example, in situ hybridization of C1q mRNA is performed together with fluorescent detection of Purkinje neuron cell bodies using Calbindin-D28K antibody. In the 2nd example, C1q mRNA in situ is performed together with 3,3’-diaminobenzidine (DAB) detection of microglia using CD68 antibody. Please note that modifications to the protocol may be needed for the use of distinct probes and antibodies, as well as alternate tissue-processing methods that are not specified herein. For appropriate examples

  6. Design of Hybrid Steam-In Situ Combustion Bitumen Recovery Processes

    SciTech Connect

    Yang Xiaomeng; Gates, Ian D.

    2009-09-15

    Given enormous capital costs, operating expenses, flue gas emissions, water treatment and handling costs of thermal in situ bitumen recovery processes, improving the overall efficiency by lowering energy requirements, environmental impact, and costs of these production techniques is a priority. Steam-assisted gravity drainage (SAGD) is the most widely used in situ recovery technique in Athabasca reservoirs. Steam generation is done on surface and consequently, because of heat losses, the energy efficiency of SAGD can never be ideal with respect to the energy delivered to the sandface. An alternative to surface steam generation is in situ combustion (ISC) where heat is generated within the formation through injection of oxygen at a sufficiently high pressure to initiate combustion of bitumen. In this manner, the heat from the combustion reactions can be used directly to mobilize the bitumen. As an alternative, the heat can be used to generate steam within the formation which then is the agent to move heat in the reservoir. In this research, alternative hybrid techniques with simultaneous and sequential steam-oxygen injection processes are examined to maximize the thermal efficiency of the recovery process. These hybrid processes have the advantage that during ISC, steam is generated within the reservoir from injected and formation water and as a product of oxidation. This implies that ex situ steam generation requirements are reduced and if there is in situ storage of combustion gases, that overall gas emissions are reduced. In this research, detailed reservoir simulations are done to examine the dynamics of hybrid processes to enable design of these processes. The results reveal that hybrid processes can lower emitted carbon dioxide-to-oil ratio by about 46%, decrease the consumed natural gas-to-oil ratio by about 73%, reduce the cumulative energy-to-oil ratio by between 40% and 70% compared to conventional SAGD, and drop water consumption per unit oil produced

  7. Tyramide Signal Amplification: Fluorescence In Situ Hybridization for Identifying Homoeologous Chromosomes.

    PubMed

    Fominaya, Araceli; Loarce, Yolanda; González, Juan M; Ferrer, Esther

    2016-01-01

    Tyramide signal amplification (TSA) fluorescence in situ hybridization (FISH) has been shown as a valuable molecular tool for visualizing specific amplified DNA sequences in chromosome preparations. This chapter describes how to perform TSA-FISH, paying special interest to its two critical steps: probe generation and metaphase plate generation. The potential of physically mapping 12S-globulin sequences by TSA-FISH as a means of identifying homeology among chromosome regions of Avena species was tested and is discussed. PMID:27511165

  8. Mapping of a rat multidrug resistance gene by fluorescence in situ hybridization

    SciTech Connect

    Popescu, N.C.; Silverman, J.A.; Thorgeirsson, S.S. )

    1993-01-01

    A cDNA clone encoding the rat mdr1b (Pgy2) gene was recently isolated and characterized. This gene has a high degree of sequence identity with other Pgy genes, particularly the mouse Pgy2 gene. By means of in situ fluorescence hybridization, the rat Pgy gene was localized on chromosome 4 band q12. This regional mapping will facilitate the identification of synteny groups on rat, mouse, and human genomes and chromosomal rearrangements during mammalian evolution. 17 refs., 2 figs.

  9. Multilocus analyses indicate a mosaic distribution of hybrid populations in ground squirrels (genus Ictidomys)

    PubMed Central

    Thompson, Cody W; Anwarali Khan, Faisal Ali; Stangl, Frederick B; Baker, Robert J; Bradley, Robert D

    2013-01-01

    DNA sequence data from mitochondrial cytochrome-b (Cytb) and Y-linked structural maintenance of chromosomes (SmcY) genes were combined with 478 nuclear loci obtained from amplified fragment length polymorphisms (AFLP) to assess the extent of hybridization and genetic spatial structure of populations in two hybridizing species of ground squirrel (Ictidomys parvidens and Ictidomys tridecemlineatus). Based on AFLP analyses of 134 individuals from 28 populations, 10 populations were identified that possessed hybrid individuals. Overall estimates of FST values revealed strong support for population structure in the Cytb data set; however, analyses of the SmcY gene and the AFLP data indicated ongoing gene flow between species. Pairwise FST comparisons of populations were not significant for the SmcY gene; although they were significant for the Cytb gene, indicating that these populations were structured and that gene flow was minimal. Therefore, gene flow between I. parvidens and I. tridecemlineatus appeared to be restricted to populations that exhibited hybridization. In addition, the fragmented nature of the geographic landscape suggested limited gene flow between populations. As a result, the distributional pattern of interspersed parental and hybrid populations were compatible with a mosaic hybrid zone model. Because ground squirrels display female philopatry and male-biased dispersal, the ecology of these species is compatible with this hypothesis. PMID:24340186

  10. A portable X-ray diffraction apparatus for in situ analyses of masters' paintings

    NASA Astrophysics Data System (ADS)

    Eveno, Myriam; Duran, Adrian; Castaing, Jacques

    2010-09-01

    It is rare that the analyses of materials in paintings can be carried out by taking micro-samples. Valuable works of art are best studied in situ by non-invasive techniques. For that purpose, a portable X-ray diffraction and fluorescence apparatus has been designed and constructed at the C2RMF. This apparatus has been used for paintings of Rembrandt, Leonardo da Vinci, Van Gogh, Mantegna, etc. Results are given to illustrate the performance of X-ray diffraction, especially when X-ray fluorescence does not bring sufficient information to conclude.

  11. Coordinated in Situ Analyses of Organic Nanoglobules in the Sutter's Mill Meteorite

    NASA Technical Reports Server (NTRS)

    Nakamura-Messenger, K.; Messenger, S.; Keller, L. P.; Clemett, S. J.; Nguyen, A. N.; Gibson, E. K.

    2013-01-01

    The Sutter's Mill meteorite is a newly fallen carbonaceous chondrite that was collected and curated quickly after its fall. Preliminary petrographic and isotopic investigations suggest affinities to the CM2 carbonaceous chondrites. The primitive nature of this meteorite and its rapid recovery provide an opportunity to investigate primordial solar system organic matter in a unique new sample. Here we report in-situ analyses of organic nanoglobules in the Sutter's Mill meteorite using UV fluorescence imaging, Fourier-transform infrared spectroscopy (FTIR), scanning transmission electron microscopy (STEM), NanoSIMS, and ultrafast two-step laser mass spectrometry (ultra-L2MS).

  12. Homoeologous chromosome pairing between the A and B genomes of Musa spp. revealed by genomic in situ hybridization

    PubMed Central

    Jeridi, Mouna; Bakry, Frédéric; Escoute, Jacques; Fondi, Emmanuel; Carreel, Françoise; Ferchichi, Ali; D'Hont, Angélique; Rodier-Goud, Marguerite

    2011-01-01

    Background and Aims Most cooking banana and several desert bananas are interspecific triploid hybrids between Musa acuminata (A genome) and Musa balbisiana (B genome). In addition, M. balbisiana has agronomical characteristics such as resistance to biotic and abiotic stresses that could be useful to improve monospecific acuminata cultivars. To develop efficient breeding strategies for improving Musa cultivars, it is therefore important to understand the possibility of chromosome exchange between these two species. Methods A protocol was developed to prepare chromosome at meiosis metaphase I suitable for genomic in situ hybridization. A series of technical challenges were encountered, the main ones being the hardness of the cell wall and the density of the microsporocyte's cytoplasm, which hampers accessibility of the probes to the chromosomes. Key parameters in solving these problems were addition of macerozyme in the enzyme mix, the duration of digestion and temperature during the spreading phase. Results and Conclusions This method was applied to analyse chromosome pairing in metaphase from triploid interspecific cultivars, and it was clearly demonstrated that interspecific recombinations between M. acuminata and M. balbisiana chromosomes do occur and may be frequent in triploid hybrids. These results provide new insight into Musa cultivar evolution and have important implications for breeding. PMID:21835815

  13. Genome-scale transcriptional analyses of first-generation interspecific sunflower hybrids reveals broad regulatory compatibility

    PubMed Central

    2013-01-01

    Background Interspecific hybridization creates individuals harboring diverged genomes. The interaction of these genomes can generate successful evolutionary novelty or disadvantageous genomic conflict. Annual sunflowers Helianthus annuus and H. petiolaris have a rich history of hybridization in natural populations. Although first-generation hybrids generally have low fertility, hybrid swarms that include later generation and fully fertile backcross plants have been identified, as well as at least three independently-originated stable hybrid taxa. We examine patterns of transcript accumulation in the earliest stages of hybridization of these species via analyses of transcriptome sequences from laboratory-derived F1 offspring of an inbred H. annuus cultivar and a wild H. petiolaris accession. Results While nearly 14% of the reference transcriptome showed significant accumulation differences between parental accessions, total F1 transcript levels showed little evidence of dominance, as midparent transcript levels were highly predictive of transcript accumulation in F1 plants. Allelic bias in F1 transcript accumulation was detected in 20% of transcripts containing sufficient polymorphism to distinguish parental alleles; however the magnitude of these biases were generally smaller than differences among parental accessions. Conclusions While analyses of allelic bias suggest that cis regulatory differences between H. annuus and H. petiolaris are common, their effect on transcript levels may be more subtle than trans-acting regulatory differences. Overall, these analyses found little evidence of regulatory incompatibility or dominance interactions between parental genomes within F1 hybrid individuals, although it is unclear whether this is a legacy or an enabler of introgression between species. PMID:23701699

  14. Detection of Hepatitis B Virus DNA in Hepatocytes, Bile Duct Epithelium, and Vascular Elements by in situ Hybridization

    NASA Astrophysics Data System (ADS)

    Blum, Hubert E.; Stowring, Linda; Figus, Annalena; Montgomery, Carolyn K.; Haase, Ashley T.; Vyas, Girish N.

    1983-11-01

    A radiolabeled probe specific for hepatitis B virus (HBV) nucleotide sequences was hybridized in situ to liver tissue from three patients with chronic hepatitis B. The HBV genome was detected not only in infected hepatocytes but also in bile duct epithelial cells, endothelial cells, and smooth muscle cells. These findings extend the known host cell range for HBV, suggest new mechanisms of viral dissemination, and illustrate the usefulness of in situ hybridization in the study of pathogenesis of HBV infection.

  15. In situ biosynthesis of bacterial nanocellulose-CaCO3 hybrid bionanocomposite: One-step process.

    PubMed

    Mohammadkazemi, Faranak; Faria, Marisa; Cordeiro, Nereida

    2016-08-01

    In this work, a simple and green route to the synthesis of the bacterial nanocellulose-calcium carbonate (BNC/CaCO3) hybrid bionanocomposites using one-step in situ biosynthesis was studied. The CaCO3 was incorporated in the bacterial nanocellulose structure during the cellulose biosynthesis by Gluconacetobacter xylinus PTCC 1734 bacteria. Hestrin-Schramm (HS) and Zhou (Z) culture media were used to the hybrid bionanocomposites production and the effect of ethanol addition was investigated. Attenuated total reflection Fourier transform infrared spectroscopy, field emission scanning electron microscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, inverse gas chromatography and thermogravimetric analysis were used to characterize the samples. The experimental results demonstrated that the ethanol and culture medium play an important role in the BNC/CaCO3 hybrid bionanocomposites production, structure and properties. The BNC/CaCO3 biosynthesized in Z culture medium revealed higher O/C ratio and amphoteric surface character, which justify the highest CaCO3 content incorporation. The CaCO3 was incorporated into the cellulosic matrix decreasing the bacterial nanocellulose crystallinity. This work reveals the high potential of in situ biosynthesis of BNC/CaCO3 hybrid bionanocomposites and opens a new way to the high value-added applications of bacterial nanocellulose. PMID:27157766

  16. In situ hybridization of oxytocin messenger RNA: macroscopic distribution and quantitation in rat hypothalamic cell groups

    SciTech Connect

    Burbach, J.P.; Voorhuis, T.A.; van Tol, H.H.; Ivell, R.

    1987-05-29

    Oxytocin mRNA was detected in the rat hypothalamus by in situ hybridization to a single stranded /sup 35/S-labelled DNA probe and the distribution of oxytocin mRNA-containing cell groups was studied at the macroscopic level. Specificity of hybridization was confirmed by comparison to vasopressin mRNA hybridization in parallel tissue sections. Cell groups containing oxytocin mRNA were confined to a set of hypothalamic cell groups, i.c. the supraoptic, paraventricular, anterior commissural nuclei, nucleus circularis and scattered hypothalamic islets. These cell groups displayed similar densities of autoradiographic signals indicating that the oxytocin gene is expressed at approximately the same average level at these various sites.

  17. In situ hybridization for the detection of hepatitis C virus RNA in human liver tissue.

    PubMed

    Li, G; Li, K; Lea, A S; Li, N L; Abdulla, N E; Eltorky, M A; Ferguson, M R

    2013-03-01

    In situ hybridization (ISH) enables visualization of specific nucleic acid in morphologically preserved cells and tissue sections. Detection of the HCV genomes in clinical specimens is useful for differential diagnosis, particularly between recurrent HCV infection and acute cellular rejection in transplant specimens. We optimized an ISH protocol that demonstrated sensitivity and specificity for detecting genomic and replicative form of HCV RNA in tissue biopsies. Digoxigenin (Dig)-labelled sense and anti-sense riboprobes were synthesized using a plasmid containing a fragment of the highly conserved HCV noncoding region as a template. The efficiency of the Dig-labelled riboprobes in detecting genomic and replicative-intermediate HCV RNA was analysed in 30 liver biopsies from patients infected or uninfected with HCV in a blinded study. A Huh7 cell line that stably replicates genome-length HCV RNA was developed to be used as a positive control. Negative control riboprobes were used in parallel to evaluate and control for background staining. The anti-sense probe detected HCV RNA in 20/21 specimens from HCV-infected liver tissues obtained from patients and in 0/9 samples from patients with non-HCV-related liver diseases, resulting in a sensitivity and specificity of 95% and 100%, respectively. HCV genomic RNA was variably distributed in tissue sections and was located primarily in the perinuclear regions in hepatocytes. Detection of HCV RNA by our optimized ISH protocol appears to be a sensitive and specific method when processing clinical specimens. It may also be revealing when exploring the pathophysiology of HCV infection by verifying the presence of viral genetic material within heptocytes and other cellular elements of diseased liver tissue. This methodology might also evaluate the response to antiviral therapies by demonstrating the absence or alteration of genetic material in clinical specimens from successfully treated patients. PMID:23383657

  18. High-throughput physical mapping of chromosomes using automated in situ hybridization.

    PubMed

    George, Phillip; Sharakhova, Maria V; Sharakhov, Igor V

    2012-01-01

    Projects to obtain whole-genome sequences for 10,000 vertebrate species and for 5,000 insect and related arthropod species are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented. Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations. Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and

  19. Automated brightfield break-apart in situ hybridization (ba-ISH) application: ALK and MALT1 genes as models.

    PubMed

    Nitta, Hiroaki; Zhang, Wenjun; Kelly, Brian D; Miller, Melanie; Pestic-Dragovich, Lidija; Bieniarz, Christopher; Vasicek, Thomas J; Marafioti, Teresa; Rimsza, Lisa; Grogan, Thomas M

    2010-12-01

    Cancer diagnosis can be a complex process, which takes consideration of histopathological, clinical, immunophenotypic, and genetic features. Since non-random chromosomal translocations are specifically involved in the development of various cancers, the detection of these gene aberrations becomes increasingly important. In recent years, break-apart (or split-signal) fluorescence in situ hybridization (FISH) has emerged as an advantageous technique to detect gene translocations on tissue sections. However, FISH assays are technically challenging and require specialized fluorescence microscopes. Furthermore, the FISH signal is not stable for long term archiving due to photo bleaching. Our objective was to demonstrate the feasibility of brightfield break-apart in situ hybridization (ba-ISH) for anaplastic lymphoma kinase (ALK) and mucosa-associated lymphoid tissue translocation protein 1 (MALT1) genes as models. ALK or MALT1 break-apart probes were labeled with digoxigenin (DIG) or 2,4-dinitrophenyl (DNP) on both sides of a known gene breakpoint region and the hybridization sites were visualized with the combination of alkaline phosphatase (AP)-based blue and red detection. Therefore, normal genes are detected as purple dots by mixing blue and red colors while translocated genes are detected as isolated blue or red dots. Formalin-fixed, paraffin-embedded tonsil was used as control for the co-localized 5' and 3' probes. Gene translocations of ALK or MALT1 were detected as separate blue and red dots on ALCL and MALT lymphoma cases. Thus, ISH analyses of gene translocations can be conducted with a regular light microscope and the long term archiving of break-apart ISH slides can be achieved. PMID:20621192

  20. Study of SGD along the French Mediterranean coastline using airborne TIR images and in situ analyses

    NASA Astrophysics Data System (ADS)

    van Beek, Pieter; Stieglitz, Thomas; Souhaut, Marc

    2015-04-01

    Although submarine groundwater discharge (SGD) has been investigated in many places of the world, very few studies were conducted along the French coastline of the Mediterranean Sea. Almost no information is available on the fluxes of water and chemical elements associated with these SGD and on their potential impact on the geochemical cycling and ecosystems of the coastal zones. In this work, we combined the use of airborne thermal infrared (TIR) images with in situ analyses of salinity, temperature, radon and radium isotopes to study SGD at various sites along the French Mediterranean coastline and in coastal lagoons. These analyses allowed us to detect SGD sites and to quantify SGD fluxes (that include both the fluxes of fresh groundwater and recirculated seawater). In particular, we will show how the Ra isotopes determined in the La Palme lagoon were used to estimate i) the residence time of waters in the lagoon and ii) SGD fluxes.

  1. Gene expression analyses in maize inbreds and hybrids with varying levels of heterosis

    PubMed Central

    Stupar, Robert M; Gardiner, Jack M; Oldre, Aaron G; Haun, William J; Chandler, Vicki L; Springer, Nathan M

    2008-01-01

    Background Heterosis is the superior performance of F1 hybrid progeny relative to the parental phenotypes. Maize exhibits heterosis for a wide range of traits, however the magnitude of heterosis is highly variable depending on the choice of parents and the trait(s) measured. We have used expression profiling to determine whether the level, or types, of non-additive gene expression vary in maize hybrids with different levels of genetic diversity or heterosis. Results We observed that the distributions of better parent heterosis among a series of 25 maize hybrids generally do not exhibit significant correlations between different traits. Expression profiling analyses for six of these hybrids, chosen to represent diversity in genotypes and heterosis responses, revealed a correlation between genetic diversity and transcriptional variation. The majority of differentially expressed genes in each of the six different hybrids exhibited additive expression patterns, and ~25% exhibited statistically significant non-additive expression profiles. Among the non-additive profiles, ~80% exhibited hybrid expression levels between the parental levels, ~20% exhibited hybrid expression levels at the parental levels and ~1% exhibited hybrid levels outside the parental range. Conclusion We have found that maize inbred genetic diversity is correlated with transcriptional variation. However, sampling of seedling tissues indicated that the frequencies of additive and non-additive expression patterns are very similar across a range of hybrid lines. These findings suggest that heterosis is probably not a consequence of higher levels of additive or non-additive expression, but may be related to transcriptional variation between parents. The lack of correlation between better parent heterosis levels for different traits suggests that transcriptional diversity at specific sets of genes may influence heterosis for different traits. PMID:18402703

  2. Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription

    PubMed Central

    Carter, Bradley S.; Fletcher, Jonathan S.; Thompson, Robert C.

    2010-01-01

    The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression. PMID:20699122

  3. Using in situ hybridization and PFGE Southern hybridization to detect translocation breakpoints in a BOR/TRPS patient cell line

    SciTech Connect

    Gu, J.Z.; Sapru, M.; Smith, D.

    1994-09-01

    Branchio-oto-renal syndrome (BOR) is an autosomal dominant disorder characterized by ear malformations, cervical fistulae, hearing loss and renal abnormalities. We have integrated the Genethon YAC contig maps with additional markers in the chromosome 8q region genetically linked by a unique patient cell line. This cell line is from a patient who has both the branchio-oto-renal syndrome and tricho-rhino-phalangeal syndrome (TRPS). High resolution cytogenetics demonstrated a direct insertion of materials from 8q13.3q21.13 to 8q24.11. TRPS has been previously linked to deletions involving 8q24.11-q24.13. The rearrangement in this patient suggests that TRPS results from loss of gene function due to insertion at the 8q24.11 breakpoint and the possible location for the BOR gene is at either of the two breakpoints of 8q13.3 and 8q21.13. We have constructed cosmid contigs in 8q24.11. In situ hybridization with cosmids mapped to these locations as probes has helped to narrow down the breakpoints. Combinations of cosmids on either side or overlapping the 8q24.11 breakpoint show split signals on one chromosome 8q arm due to insertion of the materials from the proximal region. Cosmids mapped to the TRPS deletion region have been used to hybridize to pulsed field gel genomic blots of DNA from the patient cell line and detected rearranged genomic fragments. Both in situ hybridization and genomic PFGE Southern blot will be used to precisely locate the breakpoints.

  4. In situ hybridization for the study of gene expression in neuro-otologic research.

    PubMed

    Wackym, P A; Popper, P; Ward, P H; Micevych, P E

    1990-10-01

    In situ hybridization histochemistry technology was developed for future application to neuro-otologic research. This method allowed the detection of cellular mRNA in tissue sections from the temporal bone or brainstem after cRNA/mRNA hybridization. To produce specific cRNA, single-stranded 35S-labeled cRNA (complimentary to target mRNA) is transcribed from commercially available plasmid vectors. These vectors contain promotor sequences for specific synthesis of RNA, and polylinker regions that will accept cloned DNA inserts for virtually any target nucleic acid sequence of interest. The protocol used in this research was optimized for studies that included concomitant immunohistochemical evaluation. The combination of in situ hybridization and immunohistochemistry provides the only method to correlate molecular information (gene expression) with biochemical or molecular markers, such as peptides or proteins (mRNA translation products) on individual cells in the temporal bone or brainstem. Using these techniques, we examined the distribution of the neuropeptide calcitonin gene-related peptide in rat temporal bone and brainstem sections using calcitonin gene-related peptide (CGRP) antisera and CGRP cRNA probes. We used in situ hybridization histochemistry with a cRNA probe complementary to the 3'-end noncoding sequence of the alpha CGRP mRNA and immunohistochemistry with a polyclonal antibody to the (TYR)CGRP23-37 to study the distribution of CGRP mRNA and CGRP-like immunoreactivity in the central and peripheral facial nerve. Numerous motoneuron cell bodies in the facial nucleus and accessory seventh nucleus and cell bodies in the gustatory geniculate ganglion were found to contain CGRP mRNA and the CGRP peptide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1701043

  5. Quantitative analysis of chromosome in situ hybridization signal in paraffin-embedded tissue sections.

    PubMed

    Dhingra, K; Sneige, N; Pandita, T K; Johnston, D A; Lee, J S; Emami, K; Hortobagyi, G N; Hittelman, W N

    1994-06-01

    Interphase cytogenetic analysis using chromosome-specific probes is increasingly being used to detect chromosomal aberrations on paraffin-embedded tissue sections. However, quantitative analysis of the hybridization signal is confounded by the nuclear slicing that occurs during sectioning. To determine the sensitivity and accuracy of chromosome in situ hybridization for detecting numerical chromosomal aberrations on paraffin-embedded sections, in situ hybridization was performed on sections derived from mixtures of cell populations with known frequencies of numerical chromosomal aberrations and the Chromosome Index (CI) was calculated (i.e., total number of signal spots/number of nuclei counted) as a quantitative measure of chromosome copy number. The presence of 25% or more monosomic or tetrasomic cells in a given population was easily detected as a change in CI (P < 0.05). Lower degrees of polysomy could be detected as a small percentage of nuclear fragments with > 2 signal spots. The CI was not significantly influenced by a change in section thickness from 4 to 8 microM, by an increase in cell size from 478 to 986 microM3, or by the choice of detection method (fluorescence vs. conventional bright-field microscopy). Comparative analysis of touch preparations and tissue sections from the corresponding breast tumors showed that CI accurately reflects the average copy number of chromosomes in intact nuclei and may actually be superior to in situ hybridization on whole nuclei for the detection of numerical chromosomal changes in defined histologic areas. This method is thus a sensitive and accurate means of studying genetic changes in premalignant and malignant tissue, and of assessing the genetic changes associated with specific phenotypes. PMID:7924678

  6. In situ detection of freshwater fungi in an alpine stream by new taxon-specific fluorescence in situ hybridization probes.

    PubMed

    Baschien, Christiane; Manz, Werner; Neu, Thomas R; Marvanová, Ludmila; Szewzyk, Ulrich

    2008-10-01

    New rRNA-targeting oligonucleotide probes permitted the fluorescence in situ hybridization (FISH) identification of freshwater fungi in an Austrian second-order alpine stream. Based on computer-assisted comparative sequence analysis, nine taxon-specific probes were designed and evaluated by whole-fungus hybridizations. Oligonucleotide probe MY1574, specific for a wide range of Eumycota, and the genus (Tetracladium)-specific probe TCLAD1395, as well as the species-specific probes ALacumi1698 (Alatospora acuminata), TRIang322 (Tricladium angulatum), and Alongi340 (Anguillospora longissima), are targeted against 18S rRNA, whereas probes TmarchB10, TmarchC1_1, TmarchC1_2, and AlongiB16 are targeted against the 28S rRNA of Tetracladium marchalianum and Anguillospora longissima, respectively. After 2 weeks and 3 months of exposure of polyethylene slides in the stream, attached germinating conidia and growing hyphae of freshwater fungi were accessible for FISH. Growing hyphae and germinating conidia on leaves and in membrane cages were also visualized by the new FISH probes. PMID:18776035

  7. Localization of glucocorticoid receptor messenger ribonucleic acid in hippocampus of rat brain using in situ hybridization.

    PubMed

    Yang, G; Matocha, M F; Rapoport, S I

    1988-08-01

    An in situ hybridization procedure was applied to quantify glucocorticoid receptor (GR) mRNAs in the hippocampus of rat brain. Hybridization was carried out using a radiolabeled antisense probe complementary to the rat liver GR gene. The specificity of the method was validated by showing: 1) a high cellular grain density in sections hybridized with an antisense but not a sense probe; 2) agreement between the experimental and theoretical temperature at which 50% of the hybrids melted, and 3) a high signal distribution of GR mRNA in the hippocampus, a region of brain known to preferentially concentrate steroid hormones. Within the hippocampus, however, subregional differences in hybridization densities were observed. Quantitative autoradiography indicated that the average neuronal silver grain number was highest in the pyramidal cell layers of CA2 and CA4 and lowest in those of CA1 and CA3. Also, there was a significant difference in the average grain number between all of the cell fields except for that between CA2 and CA4. These results show that contiguous but neuroanatomically distinct cell fields of the hippocampus express different levels of GR transcripts, and indicate that differential regulation of GR expression occurs in subpopulations of hippocampal neurons. PMID:3211154

  8. Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH)

    PubMed Central

    Klinger, Katherine; Landes, Greg; Shook, Donna; Harvey, Robert; Lopez, Linda; Locke, Pat; Lerner, Terry; Osathanondh, Rapin; Leverone, Benjamin; Houseal, Timothy; Pavelka, Karen; Dackowski, William

    1992-01-01

    Herein we report the results of the first major prospective study directly comparing aneuploidy detection by fluorescence in situ hybridization of interphase nuclei with the results obtained by cytogenetic analysis. We constructed probes derived from specific subregions of human chromosomes 21, 18, 13, X, and Y that give a single copy–like signal when used in conjunction with suppression hybridization. A total of 526 independent amniotic fluid samples were analyzed in a blind fashion. All five probes were analyzed on 117 samples, while subsets of these five probes were used on the remaining samples (because of insufficient sample size), for a total of over 900 autosomal hybridization reactions and over 400 sex chromosome hybridization reactions. In this blind series, 21 of 21 abnormal samples were correctly identified. The remaining samples were correctly classified as disomic for these five chromosomes. The combination of chromosome-specific probe sets composed primarily of cosmid contigs and optimized hybridization/detection allowed accurate chromosome enumeration in uncultured human amniotic fluid cells, consistent with the results obtained by traditional cytogenetic analysis. Imagesp[60]-aFigure 1 PMID:1609805

  9. Localization of glucocorticoid receptor messenger ribonucleic acid in hippocampus of rat brain using in situ hybridization

    SciTech Connect

    Yang, G.; Matocha, M.F.; Rapoport, S.I.

    1988-08-01

    An in situ hybridization procedure was applied to quantify glucocorticoid receptor (GR) mRNAs in the hippocampus of rat brain. Hybridization was carried out using a radiolabeled antisense probe complementary to the rat liver GR gene. The specificity of the method was validated by showing: 1) a high cellular grain density in sections hybridized with an antisense but not a sense probe; 2) agreement between the experimental and theoretical temperature at which 50% of the hybrids melted, and 3) a high signal distribution of GR mRNA in the hippocampus, a region of brain known to preferentially concentrate steroid hormones. Within the hippocampus, however, subregional differences in hybridization densities were observed. Quantitative autoradiography indicated that the average neuronal silver grain number was highest in the pyramidal cell layers of CA2 and CA4 and lowest in those of CA1 and CA3. Also, there was a significant difference in the average grain number between all of the cell fields except for that between CA2 and CA4. These results show that contiguous but neuroanatomically distinct cell fields of the hippocampus express different levels of GR transcripts, and indicate that differential regulation of GR expression occurs in subpopulations of hippocampal neurons.

  10. QUANTITATIVE IMAGING AND STATISTICAL ANALYSIS OF FLUORESCENCE IN SITU HYBRIDIZATION (FISH) OF AUREOBASIDIUM PULLULANS. (R823845)

    EPA Science Inventory

    Abstract

    Image and multifactorial statistical analyses were used to evaluate the intensity of fluorescence signal from cells of three strains of A. pullulans and one strain of Rhodosporidium toruloides, as an outgroup, hybridized with either a universal o...

  11. Rapid, high-resolution in situ hybridization histochemistry with radioiodinated synthetic oligonucleotides

    SciTech Connect

    Lewis, M.E.; Arentzen, R.; Baldino, F. Jr.

    1986-01-01

    In situ hybridization histochemistry is a valuable technique for localizing specific messenger RNA (mRNA) and detecting changes in gene expression. Generally, the mRNA of interest has been detected by probes obtained from cloned DNA and labelled to high specific activity by nick translation. Such probes have a number of disadvantages which can be circumvented by the use of short synthetic oligonucleotides designed to be complementary to a known mRNA sequence. We report here that synthetic oligonucleotides complementary to part of the mRNA coding for rat arginine-vasopressin (AVP) can be labelled to high specific activity with (/sup 125/I), using either the primer extension method with the Klenow fragment of DNA polymerase I or the 3'-tailing method with terminal deoxynucleotidyl transferase. Both AVP probes hybridized well to the magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. A strong autoradiographic signal was present by 2 days, with grains largely confined to the perikaryon. These results compare favorably to those obtained with (/sup 32/P)- or (/sup 3/H)-labelled probes. Given the ease of the 3'-tailing method, (/sup 125/I)-labelled oligonucleotides appear to be especially useful probes for in situ hybridization histochemistry.

  12. Fluorescent In Situ Hybridization to Detect Transgene Integration into Plant Genomes

    NASA Astrophysics Data System (ADS)

    Schwarzacher, Trude

    Fluorescent chromosome analysis technologies have advanced our understanding of genome organization during the last 30 years and have enabled the investigation of DNA organization and structure as well as the evolution of chromosomes. Fluorescent chromosome staining allows even small chromosomes to be visualized, characterized by their composition and morphology, and counted. Aneuploidies and polyploidies can be established for species, breeding lines, and individuals, including changes occurring during hybridization or tissue culture and transformation protocols. Fluorescent in situ hybridization correlates molecular information of a DNA sequence with its physical location on chromosomes and genomes. It thus allows determination of the physical position of sequences and often is the only means to determine the abundance and distribution of DNA sequences that are difficult to map with any other molecular method or would require segregation analysis, in particular multicopy or repetitive DNA. Equally, it is often the best way to establish the incorporation of transgenes, their numbers, and physical organization along chromosomes. This chapter presents protocols for probe and chromosome preparation, fluorescent in situ hybridization, chromosome staining, and the analysis of results.

  13. In-situ generation of carrier gases for scientific analyses on Mars

    NASA Technical Reports Server (NTRS)

    Finn, J. E.; Sridhar, K. R.

    1997-01-01

    The search for useful raw materials on planetary surfaces will involve various scientific analyses of soil and rock samples. The devices performing these measurements often require inert carrier gases for moving analytes and purging instrumentation. At present, the carrier or sweep gas must be carried from Earth in a compressed gas cylinder, and so the supply of this depletable resource sets a hard limit on the (flexible) life span of the experiment. If a suitable carrier gas could be produced in-situ, then the scientific return of exploration missions could be extended and enhanced greatly. Many more samples could be analyzed, long-ranging rovers could have independent gas supplies, and designs could have added flexibility with respect to gas consumption.

  14. In situ analyses on negative ions in the indium-gallium-zinc oxide sputtering process

    SciTech Connect

    Jia, Junjun; Torigoshi, Yoshifumi; Shigesato, Yuzo

    2013-07-01

    The origin of negative ions in the dc magnetron sputtering process using a ceramic indium-gallium-zinc oxide target has been investigated by in situ analyses. The observed negative ions are mainly O{sup -} with energies corresponding to the target voltage, which originates from the target and barely from the reactive gas (O{sub 2}). Dissociation of ZnO{sup -}, GaO{sup -}, ZnO{sub 2}{sup -}, and GaO{sub 2}{sup -} radicals also contributes to the total negative ion flux. Furthermore, we find that some sputtering parameters, such as the type of sputtering gas (Ar or Kr), sputtering power, total gas pressure, and magnetic field strength at the target surface, can be used to control the energy distribution of the O{sup -} ion flux.

  15. Molecular Genetic Analyses of Mating Pheromones Reveal Intervariety Mating or Hybridization in Cryptococcus neoformans

    PubMed Central

    Chaturvedi, Vishnu; Fan, Jinjiang; Stein, Birgit; Behr, Melissa J.; Samsonoff, William A.; Wickes, Brian L.; Chaturvedi, Sudha

    2002-01-01

    The sexual mating of the pathogenic yeast Cryptococcus neoformans is important for pathogenesis studies because the fungal virulence is linked to the α mating type (MATα). We characterized C. neoformans mating pheromones (MFα 1 and MFa1) from 122 strains to understand intervariety hybridization or mating and intervariety virulence. MFα 1 in three C. neoformans varieties showed (a) specific nucleotide polymorphisms, (b) different copy numbers and chromosomal localizations, and (c) unique deduced amino acids in two geographic populations of C. neoformans var. gattii. MFα 1 of different varieties cross-hybridized in Southern hybridizations. Their phylogenetic analyses showed purifying selection (neutral evolution). These observations suggested that MATα strains from any of the three C. neoformans varieties could mate or hybridize in nature with MATa strains of C. neoformans var. neoformans. A few serotype A/D diploid strains provided evidence for mating or hybridization, while a majority of A/D strains tested positive for haploid MFα 1 identical to that of C. neoformans var. grubii. MFα 1 sequence and copy numbers in diploids were identical to those of C. neoformans var. grubii, while their MFa1 sequences were identical to those of C. neoformans var. neoformans; thus, these strains were hybrids. The mice survival curves and histological lesions revealed A/D diploids to be highly pathogenic, with pathogenicity levels similar to that of the C. neoformans var. grubii type strain and unlike the low pathogenicity levels of C. neoformans var. neoformans strains. In contrast to MFα 1 in three varieties, MFa1 amplicons and hybridization signals could be obtained only from two C. neoformans var. neoformans reference strains and eight A/D diploids. This suggested that a yet undiscovered MFa pheromone(s) in C. neoformans var. gattii and C. neoformans var. grubii is unrelated to, highly divergent from, or rarer than that in C. neoformans var. neoformans. These

  16. Making a Hybrid Microfluidic Platform Compatible for In Situ Imaging by Vacuum-Based Techniques

    SciTech Connect

    Yang, Li; Yu, Xiao-Ying; Zhu, Zihua; Thevuthasan, Suntharampillai; Cowin, James P.

    2011-10-26

    A self-contained microfluidic-based device was designed and fabricated for in situ imaging of aqueous surfaces using vacuum techniques. The device is a hybrid between a microfluidic PDMS block and external accessories, all portable on a small platform (10 cm-8 cm). The key feature is that a small aperture with a diameter of 2-3 micrometers is opened to the vacuum, which serves as a detection window for in situ imaging of aqueous surfaces. Vacuum compatibility and temperature drop due to water vaporization are the two most important challenges in this invention. Theoretical calculations and fabrication strategies are presented from multiple design aspects. In addition, results from the time-of-flight secondary ion mass spectrometry (ToF-SIMS) of aqueous surfaces are presented.

  17. Reconfigurable hybrid interface for molecular marker diagnostics and in-situ reporting.

    PubMed

    Ehrhardt, Kristina; Guinn, Michael T; Quarton, Tyler; Zhang, Michael Q; Bleris, Leonidas

    2015-12-15

    Combinations of molecular signals such as transcription factors and microRNAs in cells are a reliable indicator of multi-gene disorders. A system capable of detecting these conditions in-situ may be used as a tool for diagnosis and monitoring of disease. Here, we engineer genetic circuits that sense endogenous levels of the androgen receptor (AR), the glucocorticoid receptor (GR), and the microRNA hsa-miR-21 (miR-21) in cervical cancer cells (HeLa). Furthermore, using the mediator molecule human chorionic gonadotropin (hCG), we interface the intracellular information to enzyme-linked immunosorbent assay (ELISA) test strips. We demonstrate that this hybrid genetic circuit and test-strip interface can accommodate combinatorial, low-cost, and in-situ reporting, a versatile profiling tool. PMID:26210472

  18. Localization of insulin receptor mRNA in rat brain by in situ hybridization

    SciTech Connect

    Marks, J.L.; Porte, D. Jr.; Stahl, W.L.; Baskin, D.G. )

    1990-12-01

    Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three {sup 35}S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus.

  19. Nerve growth factor mRNA in brain: localization by in situ hybridization

    SciTech Connect

    Rennert, P.D.; Heinrich, G.

    1986-07-31

    Nerve Growth Factor is a 118 amino acid polypeptide that plays an important role in the differentiation and survival of neurons. The recent discovery that a mRNA that encodes beta Nerve Growth Factor is present in brain suggests that the Nerve Growth Factor gene may not only regulate gene expression of peripheral but also of central neurons. To identify the site(s) of Nerve Growth Factor mRNA production in the brain and to determine which cells express the Nerve Growth Factor gene, the technique of in situ hybridization was employed. A 32P-labeled RNA probe complementary to Nerve Growth Factor mRNA hybridized to cells in the stratum granulosum of the dentate gyrus and the stratum pyramidale of the hippocampus. These observations identify for the first time cellular sites of Nerve Growth Factor gene expression in the central nervous system, and suggest that Nerve Growth Factor mRNA is produced by neurons.

  20. Detection of white spot syndrome virus (WSSV) of Penaeus chinensis by in situ hybridization

    NASA Astrophysics Data System (ADS)

    Zhan, Wen-Bin; Wang, Yuan-Hong; Zhang, Zhi-Dong; Hideo, Fukuda

    2000-09-01

    White Spot Syndrome Virus (WSSV) was purified from hemolymph of infected shrimp. After nucleic acid extraction from the purified virus particles, EcoR I-digested fragments of the WSSV genome were cloned; three of these fragments were used as non-radioactive probes labeled with DIG-11-dUTP. The probes hybridized in situ, with sections located in the nuclei of all WSSV-infected tissues. The virus was detected in the gill, stomach, epidermis, and connective tissue and so on, but not detected in healthy shrimp tissues and epithelial cells of hepatopancreatic tubules of diseased shrimp.

  1. Multiplexed miRNA Fluorescence In Situ Hybridization for Formalin-Fixed Paraffin-Embedded Tissues

    PubMed Central

    Renwick, Neil; Cekan, Pavol; Bognanni, Claudia; Tuschl, Thomas

    2015-01-01

    Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections. PMID:25218385

  2. Catalyzed Reported Deposition-Fluorescence In Situ Hybridization Protocol To Evaluate Phagotrophy in Mixotrophic Protists

    PubMed Central

    Medina-Sánchez, Juan M.; Felip, Marisol; Casamayor, Emilio O.

    2005-01-01

    We describe a catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH) protocol particularly suited to assess the phagotrophy of mixotrophic protists on prokaryotes, since it maintains cell and plastid integrity, avoids cell loss and egestion of prey, and allows visualization of labeled prey against plastid autofluorescence. This protocol, which includes steps such as Lugol's-formaldehyde-thiosulfate fixation, agarose cell attachment, cell wall permeabilization with lysozyme plus achromopeptidase, and signal amplification with Alexa-Fluor 488, allowed us to detect almost 100% of planktonic prokaryotes (Bacteria and Archaea) and, for the first time, to show archaeal cells ingested by mixotrophic protists. PMID:16269774

  3. In-situ silica incorporated carboxymethyl tamarind: development and application of a novel hybrid nanocomposite.

    PubMed

    Pal, Sagar; Gorain, M K; Giri, A; Bandyopadhyay, A; Panda, A B

    2011-12-01

    A novel hybrid nanocomposite has been prepared using in situ incorporation of nano-sized filler (silica) onto carboxymethyl tamarind kernel polysaccharide (CMT). Various characterizations were employed to confirm that silica nano particles have been incorporated onto the polymer matrix. Rheological characteristics reveal stronger CMT-Si interaction at 0.5 and 1 wt% level. Beyond 1 wt% Si concentration, the interaction is less and so there is little drop in shear viscosity. Flocculation efficiency increases with incorporation of nano filler, maximum efficacy being observed at 1 wt% silica concentration. All the nanocomposites exhibited better flocculation characteristics in comparison to pure CMT. PMID:21946078

  4. In situ pneumococcal vaccine production and delivery through a hybrid biological-biomaterial vector

    PubMed Central

    Li, Yi; Beitelshees, Marie; Fang, Lei; Hill, Andrew; Ahmadi, Mahmoud Kamal; Chen, Mingfu; Davidson, Bruce A.; Knight, Paul; Smith, Randall J.; Andreadis, Stelios T.; Hakansson, Anders P.; Jones, Charles H.; Pfeifer, Blaine A.

    2016-01-01

    The type and potency of an immune response provoked during vaccination will determine ultimate success in disease prevention. The basis for this response will be the design and implementation of antigen presentation to the immune system. Whereas direct antigen administration will elicit some form of immunological response, a more sophisticated approach would couple the antigen of interest to a vector capable of broad delivery formats and designed for heightened response. New antigens associated with pneumococcal disease virulence were used to test the delivery and adjuvant capabilities of a hybrid biological-biomaterial vector consisting of a bacterial core electrostatically coated with a cationic polymer. The hybrid design provides (i) passive and active targeting of antigen-presenting cells, (ii) natural and multicomponent adjuvant properties, (iii) dual intracellular delivery mechanisms, and (iv) a simple formulation mechanism. In addition, the hybrid format enables device-specific, or in situ, antigen production and consolidation via localization within the bacterial component of the vector. This capability eliminates the need for dedicated antigen production and purification before vaccination efforts while leveraging the aforementioned features of the overall delivery device. We present the first disease-specific utilization of the vector toward pneumococcal disease highlighted by improved immune responses and protective capabilities when tested against traditional vaccine formulations and a range of clinically relevant Streptococcus pneumoniae strains. More broadly, the results point to similar levels of success with other diseases that would benefit from the production, delivery, and efficacy capabilities offered by the hybrid vector. PMID:27419235

  5. A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis.

    PubMed

    Jackson, Daniel J; Herlitze, Ines; Hohagen, Jennifer

    2016-01-01

    Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a 'whole mount' tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature). PMID:27023483

  6. Multicolor fluorescence in situ hybridization and comparative genomic hybridization reveal molecular events in lung adenocarcinomas and squamous cell lung carcinomas.

    PubMed

    Shen, Hua; Gao, Wen; Wu, Yu-jie; Qiu, Hai-rong; Shu, Yong-qian

    2009-07-01

    We have used the molecular cytogenetic techniques of multicolor fluorescence in situ hybridization (M-FISH) and comparative genomic hybridization (CGH) to analyze two established lung cancer cell lines (A549, H520), 80 primary lung adenocarcinoma samples and 80 squamous cell lung carcinoma samples in order to identify common chromosomal aberrations. M-FISH revealed numerous complex chromosomal rearrangements. Chromosomes 5, 6, 11, 12, and 17 were most frequently involved in interchromosomal translocations. CGH revealed regions on 1q, 2p, 3q, 5p, 5q, 7p, 8q, 11q, 12q, 14q, 16p, 17p, 19q, 20q, 21q and 22q to be commonly over-represented and regions on 2q, 3p, 4p, 5q, 7q, 8p, 9p, 13q, 14q, and 17p to be under-represented. In lung adenocarcinomas the most common gains were found in 16p13 (50%); while in squamous cell lung carcinomas the common gains were found in 17q21 (45%) and these alterations were observed to be associated with their specific pathological subtype. In conclusion, the present study contributes to the molecular biological characterization in lung adenocarcinomas and squamous cell lung carcinomas and through evaluation of molecular events to the recently emergent focus on novel markers for lung cancer treatment. PMID:18848758

  7. Resolution-improved in situ DNA hybridization detection based on microwave photonic interrogation.

    PubMed

    Cao, Yuan; Guo, Tuan; Wang, Xudong; Sun, Dandan; Ran, Yang; Feng, Xinhuan; Guan, Bai-ou

    2015-10-19

    In situ bio-sensing system based on microwave photonics filter (MPF) interrogation method with improved resolution is proposed and experimentally demonstrated. A microfiber Bragg grating (mFBG) is used as sensing probe for DNA hybridization detection. Different from the traditional wavelength monitoring technique, we use the frequency interrogation scheme for resolution-improved bio-sensing detection. Experimental results show that the frequency shift of MPF notch presents a linear response to the surrounding refractive index (SRI) change over the range of 1.33 to 1.38, with a SRI resolution up to 2.6 × 10(-5) RIU, which has been increased for almost two orders of magnitude compared with the traditional fundamental mode monitoring technique (~3.6 × 10(-3) RIU). Due to the high Q value (about 27), the whole process of DNA hybridization can be in situ monitored. The proposed MPF-based bio-sensing system provides a new interrogation method over the frequency domain with improved sensing resolution and rapid interrogation rate for biochemical and environmental measurement. PMID:26480367

  8. Gene numerical imbalances in cytological specimens based on fluorescence/chromogenic in situ hybridization analysis.

    PubMed

    Tsiambas, E; Karameris, A; Lygeros, M; Athanasiou, A E; Salemis, N S; Gourgiotis, S; Ragkos, V; Metaxas, G E; Vilaras, G; Patsouris, E

    2012-01-01

    Design and development of novel targeted therapeutic strategies is an innovation in handling patients with solid malignancies including breast, colon, lung, head & neck or even pancreatic and hepatocellular carcinoma. For a long time, immunohistocytochemistry (IHC/ICC) has been performed as a routine method in almost all labs for evaluating protein expression. Modern molecular approaches show that identification of specific structural and numerical imbalances regarding genes involved in signal transduction pathways provide important data to the oncologists. Alterations in molecules such as epidermal growth factor receptor (EGFR), HER2/neu, PTEN or Topoisomerase IIa affect the response rates to specific chemotherapeutic agents modifying also patients' prognostic rates. In situ hybridization (ISH) techniques based on fluorescence and chromogenic variants (FISH/CISH) or silver in situ hybridization (SISH) are applicable in both tissue and cell substrates. Concerning cytological specimens, FISH/CISH analysis appears to be a fast and very accurate method in estimating gene/chromosome ratios. In this paper, we sought to evaluate the usefulness of FISH/ CISH analysis in cytological specimens, describing also the advantages and disadvantages of these methods from the technical point of view. PMID:23033306

  9. Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)

    SciTech Connect

    Deutsch, Eric W.; Ball, Catherine A.; Berman, Jules J.; Bova, G. Steven; Brazma, Alvis; Bumgarner, Roger E.; Campbell, David; Causton, Helen C.; Christiansen, Jeff; Daian, Fabrice; Dauga, Delphine; Davidson, Duncan; Gimenez, Gregory; Goo, Young Ah; Grimmond, Sean; Henrich, Thorsten; Herrmann, Bernhard G.; Johnson, Michael H.; Korb, Martin; Mills, Jason C.; Oudes, Asa; Parkinson, Helen E.; Pascal, Laura E.; Pollet, Nicolas; Quackenbush, John; Ramaialison, Mirana; Ringwald, Martin; Salgado, David; Sansone, Susanna A.; Sherlock, Gavin; Stoeckert, Christian Jr. J.; Swedlow, Jason; Taylor, Ronald C.; Walasheck, Laura; Warford, Anthony; Wilkinson, David G.; Zhou, Yi; Zon, Leonard I.; Liu, Alvin Y.; True, Lawrence D.

    2008-03-28

    Herein, we present for consideration such a specification, termed “Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)”. It is modelled after the MIAME (Minimum Information About a Microarray Experiment) specification for microarray experiments. The purpose of data standards like MIAME and MISFISHIE is to specify information content without specifying a format for encoding that information. The MISFISHIE standard specifies six sections of information that must be detailed for each experiment: Experimental Design, Specimens, Reporters, Staining, Imaging Data, and Image Characterizations. A general checklist is provided to quickly and efficiently establish adherence to the standard. Currently, we estimate that most articles describing gene expression localization studies, such as in situ hybridization assays, do not fully provide the minimum information needed for independent verification of results. In a small survey of 32 journal articles from the past five years, we found that nearly 90% did not meet all the requirements, although many met most of them. We propose that requiring authors to provide the minimum experimental detail about gene expression localization experiments would substantially facilitate reproducibility and interpretability of results by fellow investigators. Furthermore, inclusion of specific experimental details such as reagents and methods in publications would ultimately allow others to readily search the literature for these data items, especially given the ongoing trend towards open access full text journals.

  10. Detection of a complex translocation using fluorescent in situ hybridization (FISH)

    SciTech Connect

    Rosen, B.A.; Abuelo, D.N.; Mark, H.F.

    1994-09-01

    The use of fluorescent in situ hybridization (FISH) allowed the detection of a complex 3-way translocation in a patient with multiple congenital malformations and mental retardation. The patient was a 10-year-old girl with mental retardation, seizures, repaired cleft palate, esotropia, epicanthal folds, broad nasal bridge, upward slanting palpebral fissures, single transverse palmar crease, brachydactyly, hypoplastic nails, ectrodactyly between the third and fourth right toes, and hypoplasia of the left third toe. Chromosome analysis performed at birth was reported as normal. We performed high resolution banding analysis which revealed an apparently balanced translocation between chromosomes 2 and 9. However, because of her multiple abnormalities, further studies were ordered. Fluorescent in situ hybridization (FISH) using chromosome painting probes revealed a karyotype of 46,XX,t(2;8;9) (2pter{yields}q31::8q21.2{yields}8qter; 8pter{yields}q21.2::2q31{yields}q34::9q34{yields}qter; 9pter{yields}q34::2q34{yields}qter). The 3-way translocation appears to be de novo, as neither parent is a translocation carrier. This case illustrates the importance of using FISH to further investigate cases of apparently balanced translocations in the presence of phenotypic abnormalities and/or mental retardation.

  11. Confocal Raman microscopy and fluorescent in situ hybridization - A complementary approach for biofilm analysis.

    PubMed

    Kniggendorf, Ann-Kathrin; Nogueira, Regina; Kelb, Christian; Schadzek, Patrik; Meinhardt-Wollweber, Merve; Ngezahayo, Anaclet; Roth, Bernhard

    2016-10-01

    We combine confocal Raman microscopy (CRM) of wet samples with subsequent Fluorescent in situ hybridization (FISH) without significant limitations to either technique for analyzing the same sample of a microbial community on a cell-to-cell basis. This combination of techniques allows a much deeper, more complete understanding of complex environmental samples than provided by either technique alone. The minimalistic approach is based on laboratory glassware with micro-engravings for reproducible localization of the sample at cell scale combined with a fixation and de- and rehydration protocol for the respective techniques. As proof of concept, we analyzed a floc of nitrifying activated sludge, demonstrating that the sample can be tracked with cell-scale precision over different measurements and instruments. The collected information includes the microbial content, spatial shape, variant chemical compositions of the floc matrix and the mineral microparticles embedded within. In addition, the direct comparison of CRM and FISH revealed a difference in reported cell size due to the different cell components targeted by the respective technique. To the best of our knowledge, this is the first report of a direct cell-to-cell comparison of confocal Raman microscopy and Fluorescent in situ hybridization analysis performed on the same sample. An adaptation of the method to include native samples as a starting point is planned for the near future. The micro-engraving approach itself also opens up the possibility of combining other, functionally incompatible techniques as required for further in-depth investigations of low-volume samples. PMID:27423128

  12. Next-Generation in Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability

    PubMed Central

    2014-01-01

    Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability. PMID:24712299

  13. Paratuberculosis in sheep: Histochemical, immunohistochemical and in situ hybridization evidence of in utero and milk transmission.

    PubMed

    Verin, Ranieri; Perroni, Marco; Rossi, Giacomo; De Grossi, Luigi; Botta, Roberto; De Sanctis, Bruno; Rocca, Stefano; Cubeddu, Tiziana; Crosby-Durrani, Hayley; Taccini, Ennio

    2016-06-01

    To investigate in utero and milk transmission of Mycobacterium avium subsp. paratuberculosis (MAP), tissues from thirteen pregnant sheep, naturally infected and serologically positive to MAP, were examined by means of histochemistry, immunohistochemistry and in situ hybridization. Soon after parturition, ewes were euthanized and tissues samples were collected and prepared. The offspring (18 lambs) were divided into three groups to investigate different routes of MAP transmission. Lambs were sacrificed at three months old and the tissue samples collected, formalin-fixed and paraffin embedded. Hematoxylin and eosin and Ziehl-Neelsen staining methods were performed on fixed tissues for general examination and for detection of acid-fast bacteria. Additionally, immunohistochemical and in situ hybridization techniques were used to detect MAP antigen and MAP DNA respectively. This study of a flock of MAP-infected sheep indicates both in utero and milk transmission of MAP from dams to their offspring. Importantly, this study detected the presence of MAP in the mammary gland and mammary lymph nodes of adult ewes therefore indicating a significant route for the potential exposure to humans from this bacterial infection. PMID:27234557

  14. In Situ Hybridization Methods for Mouse Whole Mounts and Tissue Sections with and Without Additional β-Galactosidase Staining

    PubMed Central

    Komatsu, Yoshihiro; Kishigami, Satoshi; Mishina, Yuji

    2014-01-01

    In situ hybridization is a powerful method for detecting endogenous mRNA sequences in morphologically preserved samples. We provide in situ hybridization methods, which are specifically optimized for mouse embryonic samples as whole mounts and section tissues. Additionally, β-Galactosidase (β-gal) is a popular reporter for detecting the expression of endogenous or exogenous genes. We reveal that 6-chloro-3-indoxyl-β-D-galactopyranoside (S-gal) is a more sensitive substrate for β-gal activity than 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal). S-gal is advantageous where β-gal activity is limited including early stage mouse embryos. As a result of the increased sensitivity as well as the color compatibility of S-gal, we successfully combined β-gal staining using S-gal with in situ hybridization using DIG-labeled probes in both whole mounts and sections. PMID:24318810

  15. Development of automated brightfield double In Situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence In Situ hybridization (FISH)

    PubMed Central

    Nitta, Hiroaki; Hauss-Wegrzyniak, Beatrice; Lehrkamp, Megan; Murillo, Adrian E; Gaire, Fabien; Farrell, Michael; Walk, Eric; Penault-Llorca, Frederique; Kurosumi, Masafumi; Dietel, Manfred; Wang, Lin; Loftus, Margaret; Pettay, James; Tubbs, Raymond R; Grogan, Thomas M

    2008-01-01

    Background Human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas. Methods The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark® XT slide processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H2O2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatise (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives. Results Sequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 – 1.0000) and the ASCO

  16. An Optimized Small Tissue Handling System for Immunohistochemistry and In Situ Hybridization.

    PubMed

    Anthony, Giovanni; Lee, Ju-Ahng

    2016-01-01

    Recent development in 3D printing technology has opened an exciting possibility for manufacturing 3D devices on one's desktop. We used 3D modeling programs to design 3D models of a tissue-handling system and these models were "printed" in a stereolithography (SLA) 3D printer to create precision histology devices that are particularly useful to handle multiple samples with small dimensions in parallel. Our system has been successfully tested for in situ hybridization of zebrafish embryos. Some of the notable features include: (1) A conveniently transferrable chamber with 6 mesh-bottomed wells, each of which can hold dozens of zebrafish embryos. This design allows up to 6 different samples to be treated per chamber. (2) Each chamber sits in a well of a standard 6-well tissue culture plate. Thus, up to 36 different samples can be processed in tandem using a single 6 well plate. (3) Precisely fitting lids prevent solution evaporation and condensation, even at high temperatures for an extended period of time: i.e., overnight riboprobe hybridization. (4) Flat bottom mesh maximizes the consistent treatment of individual tissue samples. (5) A magnet-based lifter was created to handle up to 6 chambers (= 36 samples) in unison. (6) The largely transparent resin aids in convenient visual inspection both with eyes and using a stereomicroscope. (7) Surface engraved labeling enables an accurate tracking of different samples. (8) The dimension of wells and chambers minimizes the required amount of precious reagents. (9) Flexible parametric modeling enables an easy redesign of the 3D models to handle larger or more numerous samples. Precise dimensions of 3D models and demonstration of how we use our devices in whole mount in situ hybridization are presented. We also provide detailed information on the modeling software, 3D printing tips, as well as 3D files that can be used with any 3D printer. PMID:27489962

  17. c-myc copy number gains in bladder cancer detected by fluorescence in situ hybridization.

    PubMed Central

    Sauter, G.; Carroll, P.; Moch, H.; Kallioniemi, A.; Kerschmann, R.; Narayan, P.; Mihatsch, M. J.; Waldman, F. M.

    1995-01-01

    Amplification and overexpression of c-myc have been suggested as prognostic markers in human cancer. To assess the role of c-myc gene copy number alterations in bladder cancer, 87 bladder tumors were examined for c-myc aberrations by fluorescence in situ hybridization. Dual labeling hybridization with a repetitive pericentromeric probe specific for chromosome 8 and a probe for the c-myc locus (at 8q24) was performed to analyze c-myc copy number in relation to chromosome 8 copy number on a cell by cell basis. A clear-cut c-myc amplification (up to 40 to 150 copies per cell) was found in 3 tumors. There was a low level c-myc copy number increase in 32 of the remaining 84 tumors. There was no association of low level c-myc copy number increase with c-myc protein overexpression. This suggests that a c-myc gene copy number gain as detected by fluorescence in situ hybridization does not necessarily reflect a disturbed c-myc gene function but may indicate a structural chromosome 8 abnormality including gain of distal 8q. The strong association of low level c-myc (8q) gains with tumor grade (P < 0.0001), stage (P < 0.0001), chromosome polysomy (P < 0.0001), p53 protein expression (P = 0.0019), p53 deletion (P = 0.0403), and tumor cell proliferation (Ki67 labeling index; P = 0.0021) is consistent with a role of chromosome 8 alterations in bladder cancer progression. Images Figure 1 PMID:7747807

  18. An Optimized Small Tissue Handling System for Immunohistochemistry and In Situ Hybridization

    PubMed Central

    Anthony, Giovanni; Lee, Ju-Ahng

    2016-01-01

    Recent development in 3D printing technology has opened an exciting possibility for manufacturing 3D devices on one’s desktop. We used 3D modeling programs to design 3D models of a tissue-handling system and these models were “printed” in a stereolithography (SLA) 3D printer to create precision histology devices that are particularly useful to handle multiple samples with small dimensions in parallel. Our system has been successfully tested for in situ hybridization of zebrafish embryos. Some of the notable features include: (1) A conveniently transferrable chamber with 6 mesh-bottomed wells, each of which can hold dozens of zebrafish embryos. This design allows up to 6 different samples to be treated per chamber. (2) Each chamber sits in a well of a standard 6-well tissue culture plate. Thus, up to 36 different samples can be processed in tandem using a single 6 well plate. (3) Precisely fitting lids prevent solution evaporation and condensation, even at high temperatures for an extended period of time: i.e., overnight riboprobe hybridization. (4) Flat bottom mesh maximizes the consistent treatment of individual tissue samples. (5) A magnet-based lifter was created to handle up to 6 chambers (= 36 samples) in unison. (6) The largely transparent resin aids in convenient visual inspection both with eyes and using a stereomicroscope. (7) Surface engraved labeling enables an accurate tracking of different samples. (8) The dimension of wells and chambers minimizes the required amount of precious reagents. (9) Flexible parametric modeling enables an easy redesign of the 3D models to handle larger or more numerous samples. Precise dimensions of 3D models and demonstration of how we use our devices in whole mount in situ hybridization are presented. We also provide detailed information on the modeling software, 3D printing tips, as well as 3D files that can be used with any 3D printer. PMID:27489962

  19. SUPERSENSITIVE IN SITU HYBRIDIZATION BY TYRAMIDE SIGNAL AMPLIFICATION AND NANOGOLD SILVER STAINING: THE CONTRIBUTION OF AUTOMETALLOGRAPHY AND CATALYZED REPORTER DEPOSITION TO THE REJUVENATION OF IN SITU HYBRIDIZATION.

    SciTech Connect

    TUBBS,R.R.PETTAY,J.GROGAN,T.CHEUNG,A.L.M.POWELL,R.D.HAINFELD,J.HAUSER-KRONBERGER,C.HACKER,G.W.

    2002-04-17

    It is peculiar that in situ hybridization (ISH), a technique with many similarities to immunohistochemistry (IHC), has not enjoyed the phenomenal growth in both basic research and clinical applications as has its sister technique IHC. Since the late 1970s, when immunoperoxidase techniques began to be applied to routine diagnostic material and to numerous research applications, there has been a natural evolution of the IHC procedure. Namely, only a few primary antibodies were available commercially at the onset, and only one indirect and the peroxidase-antiperoxidase (PAP) technique detection systems were in place. With the advent of avidin-biotin detection systems and monoclonal antibodies, and a viable commercial market, extraordinary growth of the procedure's applications in clinical research and diagnostic pathology occurred during the subsequent two decades. Today, IHC is automated and widely used for research purposes and, to a large extent, has become a routine diagnostic ''special stain'' in most clinical laboratories. During the same period, ISH enjoyed very little growth in both research and diagnostic applications. What has accounted for this lack of maturation of the technique? The success of IHC is part of the reason measuring a gene's encoded protein routinely and inexpensively, particularly as automation evolved, rendered IHC a more viable choice in many instances. Inherent comparative sensitivity of the procedures has also clearly been a factor. Unfortunately, the chromogenic procedures in place are often insufficiently sensitive to detect the relatively low amounts of DNA and RNA levels at which the clinical utility is to be found.

  20. Novel fluorescence in situ hybridization approaches in solid tumors. Characterization of frozen specimens, touch preparations, and cytological preparations.

    PubMed Central

    Xiao, S.; Renshaw, A.; Cibas, E. S.; Hudson, T. J.; Fletcher, J. A.

    1995-01-01

    Fluorescence in situ hybridization has emerged as an extremely important tool for detection and characterization of nonrandom chromosome aberrations in cancer. Fluorescence in situ hybridization assays have been very reliable in cytogenetic tumor preparations, but have been more unpredictable in archival, paraffin-embedded specimens. We describe novel approaches for detection of chromosome aberrations in frozen tumor specimens, touch preparations, and cytological preparations. These approaches are both simple and reproducible, with minimal case-to-case variation in hybridization efficiency or hybridization signal quality. We demonstrate potential applications of these novel approaches by evaluating: 1) significance of normal karyotypes in malignant peripheral nerve sheath tumors; 2) p15/p16 copy number in prostate cancer; and 3) clonal chromosome 3p deletion in cytological preparations of pleural fluid from patients with mesothelioma. Images Figure 1 PMID:7573365

  1. Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models.

    PubMed

    Fontenete, Sílvia; Guimarães, Nuno; Wengel, Jesper; Azevedo, Nuno Filipe

    2016-06-01

    The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of one, two or more complementary nucleic acid strands, has been studied for many years. The appearance of the nearest-neighbor model led to several theoretical and experimental papers on DNA thermodynamics that provide reasonably accurate thermodynamic information on nucleic acid duplexes and allow estimation of the melting temperature. Because there are no thermodynamic models specifically developed to predict the hybridization temperature of a probe used in a fluorescence in situ hybridization (FISH) procedure, the melting temperature is used as a reference, together with corrections for certain compounds that are used during FISH. However, the quantitative relation between melting and experimental FISH temperatures is poorly described. In this review, various models used to predict the melting temperature for rRNA targets, for DNA oligonucleotides and for nucleic acid mimics (chemically modified oligonucleotides), will be addressed in detail, together with a critical assessment of how this information should be used in FISH. PMID:25586037

  2. Identification of two Skeletonema costatum-like diatoms by fluorescence in situ hybridization

    NASA Astrophysics Data System (ADS)

    Zhang, Baoyu; Chen, Guofu; Wang, Guangce; Lu, Douding

    2010-03-01

    A harmful algae bloom (HAB) is a dense aggregation of algae in a marine or aquatic environment that can result in significant environmental problems. To forecast the occurrence of HAB, development of a rapid and precise detection method is urgently required. In this study, two Skeletonema costatum-like diatoms (SK-1 and SK-2), were identified morphologically under a light microscope, and detected using fluorescent in situ hybridization (FISH). Strain SK-1 was isolated from a frequently HAB affected area of the East China Sea, and strain SK-2 from an aquatic farm in Qingdao, China. Fluorescent DNA probes were designed that were complementary to the ITS sequence (including 5.8S rDNA) of strain SK-1. After hybridization, strong green fluorescence was observed in cells of strain SK-1 under an epifluorescence microscope; however, no such fluorescence was observed with strain SK-2, which indicates that probes hybridized only the DNA of the target strain, SK-1, in species-specific manner, and that the two strains do not belong to a same species. This finding was confirmed by ITS sequence analysis. The FISH technique used in this study was sensitive, simple, and rapid, and is a promising tool for detecting target HAB species in natural environments.

  3. Identification of genetic changes associated with drug resistance by reverse in situ hybridization.

    PubMed Central

    Hoare, S. F.; Freeman, C. A.; Coutts, J. C.; Varley, J. M.; James, L.; Keith, W. N.

    1997-01-01

    The molecular cytogenetic techniques of comparative genomic hybridization (CGH) and reverse in situ hybridization (REVISH) allow the entire genomes of tumours to be screened for genetic changes without the requirement for specific probes or markers. In order to define the ability of REVISH to detect and map regions of amplification associated with drug resistance, we investigated a panel of cell lines selected for resistance to doxorubicin and intrinsic sensitivity to topoisomerase II-inhibitory drugs. We have defined a modified REVISH protocol, which involves double hybridizations with genomic DNA from the test cell lines and chromosome-specific whole chromosome paints to identify the chromosomes to which the amplicons localize. Sites of amplification are then mapped by fractional length measurements (Flpter), using published genome databases. Our findings show that amplification of the topoisomerase II alpha gene is readily detected and mapped, as is amplification of the MDR and MRP loci. Interestingly, REVISH detected a new amplicon in the doxorubicin-resistant lung cancer cell line, GLC4-ADR, which mapped to chromosome 1q. REVISH is therefore ideally suited to characterize genetic changes specific for drug resistance within a background of genetic anomalies associated with tumour progression. Images Figure 1 Figure 2 Figure 3 PMID:9010038

  4. Multiplex Ligation-Dependent Probe Amplification Versus Multiprobe Fluorescence in Situ Hybridization To Detect Genomic Aberrations in Chronic Lymphocytic Leukemia

    PubMed Central

    Al Zaabi, Eiman A.; Fernandez, Louis A.; Sadek, Irene A.; Riddell, D. Christie; Greer, Wenda L.

    2010-01-01

    Cytogenetic abnormalities play a major role in the prognosis of patients with chronic lymphocytic leukemia (CLL). Several methods have emerged to try to best identify these abnormalities. We used fluorescence in situ hybridization (FISH) to determine the frequency of cytogenetic changes in our CLL patient population. We also evaluated the effectiveness of multiplex ligation-dependent probe amplification (MLPA) in detecting these abnormalities. Sixty-two B-CLL patients and 20 healthy controls were enrolled, and FISH and MLPA analyses were performed on peripheral blood samples. Using FISH, genomic aberrations were found in 73% of patients and presented as follows: single 13q14.3 deletion (60%), trisomy 12 (7%), ATM deletion (6%), 17p13.1 deletion (2%). MLPA analyses done on 61/62 patients showed sensitivity and specificity values of 90% and 100% respectively. MLPA revealed several additional copy number changes, the most common being 19p13 (LDLR and CDKN2D). Moreover, the cost for MLPA analysis, including technical time and reagents, is 86% less than FISH. In conclusion, cytogenetic abnormalities are a common finding in CLL patients, and MLPA is a reliable approach that is more cost effective and faster than FISH. Despite MLPA limitations of sensitivity, it can be used as a first-line screen and complementary test to FISH analysis. PMID:20093390

  5. Simultaneous visualization of different genomes (J, JSt and St) in a Thinopyrum intermedium × Thinopyrum ponticum synthetic hybrid (Poaceae) and in its parental species by multicolour genomic in situ hybridization (mcGISH).

    PubMed

    Kruppa, Klaudia; Molnár-Láng, Márta

    2016-01-01

    Multicolour genomic in situ hybridization (mcGISH) using total genomic DNA probes from Thinopyrum bessarabicum (Săvulescu & Rayss, 1923) Á. Löve, 1984 (genome J(b) or E(b), 2n = 14), and Pseudoroegneria spicata (Pursh, 1814) Á. Löve, 1980 (genome St, 2n = 14) was used to characterize the mitotic metaphase chromosomes of a synthetic hybrid of Thinopyrum intermedium (Host, 1805) Barkworth & D.R. Dewey, 1985 and Thinopyrum ponticum (Podpěra, 1902) Z.-W. Liu et R.-C.Wang, 1993 named "Agropyron glael" and produced by N.V. Tsitsin in the former Soviet Union. The mcGISH pattern of this synthetic hybrid was compared to its parental wheatgrass species. Hexaploid Thinopyrum intermedium contained 19 J, 9 J(St) and 14 St chromosomes. The three analysed Thinopyrum ponticum accessions had different chromosome compositions: 43 J + 27 J(St) (PI531737), 40 J + 30 J(St) (VIR-44486) and 38 J + 32 J(St) (D-3494). The synthetic hybrid carried 18 J, 28 J(St) and 8 St chromosomes, including one pair of J-St translocation and/or decreased fluorescent intensity, resulting in unique hybridization patterns. Wheat line Mv9kr1 was crossed with the Thinopyrum intermedium × Thinopyrum ponticum synthetic hybrid in Hungary in order to transfer its advantageous agronomic traits (leaf rust and yellow rust resistance) into wheat. The chromosome composition of a wheat/A.glael F1 hybrid was 21 wheat + 28 wheatgrass (11 J + 14 J(St)+ 3 S). In the present study, mcGISH involving the simultaneous use of St and J genomic DNA as probes provided information about the type of Thinopyrum chromosomes in a Thinopyrum intermedium/Thinopyrum ponticum synthetic hybrid called A. glael. PMID:27551349

  6. Simultaneous visualization of different genomes (J, JSt and St) in a Thinopyrum intermedium × Thinopyrum ponticum synthetic hybrid (Poaceae) and in its parental species by multicolour genomic in situ hybridization (mcGISH)

    PubMed Central

    Kruppa, Klaudia; Molnár-Láng, Márta

    2016-01-01

    Abstract Multicolour genomic in situ hybridization (mcGISH) using total genomic DNA probes from Thinopyrum bessarabicum (Săvulescu & Rayss, 1923) Á. Löve, 1984 (genome Jb or Eb, 2n = 14), and Pseudoroegneria spicata (Pursh, 1814) Á. Löve, 1980 (genome St, 2n = 14) was used to characterize the mitotic metaphase chromosomes of a synthetic hybrid of Thinopyrum intermedium (Host, 1805) Barkworth & D.R. Dewey, 1985 and Thinopyrum ponticum (Podpěra, 1902) Z.-W. Liu et R.-C.Wang, 1993 named „Agropyron glael” and produced by N.V. Tsitsin in the former Soviet Union. The mcGISH pattern of this synthetic hybrid was compared to its parental wheatgrass species. Hexaploid Thinopyrum intermedium contained 19 J, 9 JSt and 14 St chromosomes. The three analysed Thinopyrum ponticum accessions had different chromosome compositions: 43 J + 27 JSt (PI531737), 40 J + 30 JSt (VIR-44486) and 38 J + 32 JSt (D-3494). The synthetic hybrid carried 18 J, 28 JSt and 8 St chromosomes, including one pair of J-St translocation and/or decreased fluorescent intensity, resulting in unique hybridization patterns. Wheat line Mv9kr1 was crossed with the Thinopyrum intermedium × Thinopyrum ponticum synthetic hybrid in Hungary in order to transfer its advantageous agronomic traits (leaf rust and yellow rust resistance) into wheat. The chromosome composition of a wheat/A.glael F1 hybrid was 21 wheat + 28 wheatgrass (11 J + 14 JSt+ 3 S). In the present study, mcGISH involving the simultaneous use of St and J genomic DNA as probes provided information about the type of Thinopyrum chromosomes in a Thinopyrum intermedium/Thinopyrum ponticum synthetic hybrid called A. glael. PMID:27551349

  7. Coordinated In Situ Analyses of Organic Nanoglobules in the Sutter's Mill Meteorite

    NASA Technical Reports Server (NTRS)

    Nakamura--Messenger, K.; Messenger, S.; Keller, L. P.; Clemett, S. J.; Nguyen, A. N.; Gibson, E. K.

    2013-01-01

    The Sutter s Mill meteorite is a newly fallen carbonaceous chondrite that was collected and curated quickly after its fall [1]. Preliminary petrographic and isotopic investigations suggest affinities to the CM2 carbonaceous chondrites. The primitive nature of this meteorite and its rapid recovery provide an opportunity to investigate primordial solar system organic matter in a unique new sample. Organic matter in primitive meteorites and chondritic porous interplanetary dust particles (CP IDPs) is commonly enriched in D/H and N-15/N-14 relative to terrestrial values [2-4]. These anomalies are ascribed to the partial preservation of presolar cold molecular cloud material [2]. Some meteorites and IDPs contain gm-size inclusions with extreme H and N isotopic anomalies [3-5], possibly due to preserved primordial organic grains. The abundance and isotopic composition of C in Sutter's Mill were found to be similar to the Tagish Lake meteorite [6]. In the Tagish Lake meteorite, the principle carriers of large H and N isotopic anomalies are sub-micron hollow organic spherules known as organic nanoglobules [7]. Organic nanoglobules are commonly distributed among primitive meteorites [8, 9] and cometary samples [10]. Here we report in-situ analyses of organic nano-globules in the Sutter's Mill meteorite using UV fluorescence imaging, Fourier-transform infrared spectroscopy (FTIR), scanning transmission electron microscopy (STEM), NanoSIMS, and ultrafast two-step laser mass spectrometry (ultra-L2MS).

  8. Portable apparatus for in situ x-ray diffraction and fluorescence analyses of artworks.

    PubMed

    Eveno, Myriam; Moignard, Brice; Castaing, Jacques

    2011-10-01

    A portable X-ray fluorescence/X-ray diffraction (XRF/XRD) system for artwork studies has been designed constructed and tested. It is based on Debye Scherrer XRD in reflection that takes advantage of many recent improvements in the handling of X-rays (polycapillary optics; advanced two-dimensional detection). The apparatus is based on a copper anode air cooled X-ray source, and the XRD analysis is performed on a 5-20 μm thick layer from the object surface. Energy dispersive XRF elemental analysis can be performed at the same point as XRD, giving elemental compositions that support the interpretation of XRD diagrams. XRF and XRD analyses were tested to explore the quality and the limits of the analytical technique. The XRD diagrams are comparable in quality with diagrams obtained with conventional laboratory equipment. The mineral identification of materials in artwork is routinely performed with the portable XRF-XRD system. Examples are given for ceramic glazes containing crystals and for paintings where the determination of pigments is still a challenge for nondestructive analysis. For instance, lead compounds that provide a variety of color pigments can be easily identified as well as a pigment such as lapis lazuli that is difficult to identify by XRF alone. More than 70 works of art have been studied in situ in museums, monuments, etc. In addition to ceramics and paintings, these works include bronzes, manuscripts, etc., which permit improvement in the comprehension of ancient artistic techniques. PMID:21615981

  9. Dating the longevity of ductile shear zones: Insight from 40Ar/39Ar in situ analyses

    NASA Astrophysics Data System (ADS)

    Schneider, Susanne; Hammerschmidt, Konrad; Rosenberg, Claudio L.

    2013-05-01

    We attempt to improve temporal constraints on the longevity and the termination of ductile shear zones by performing texturally-controlled in situ 40Ar/39Ar analyses of pre-kinematic muscovite, biotite and K-feldspars, of syn-kinematic phengite and K-feldspar, and of post-kinematic phengite within the same samples of sinistral shear zones from the western Tauern Window (Eastern Alps). Additionally two samples were dated by the Rb/Sr method (microsampling). Relative sequences of mineral formation based on microstructural, cross-cutting relationships were confirmed by in situ 40Ar/39Ar analyses, showing that syn-kinematic minerals are, in general, younger than pre-kinematic minerals and older or of equal age than the post-kinematic minerals of the same sample. From the rim to the core of the western Tauern Window syn-kinematic phengite and K-feldspar reveal a set of formation ages varying between 33 and 15 Ma for the northernmost and peripheral shear zone (Ahorn Shear Zone), between 24 and 12 Ma for the intermediate shear zone network (Tuxer Shear Zones), and between 20 and 7 Ma for the southernmost and central shear zone (Greiner Shear Zone). The age variation of syn-kinematic phengite and K-feldspar analyses is larger than the analytical error of each age obtained. In addition, isochron calculations of the syn-kinematic minerals reveal atmospheric-like 40Ar/36Ar intercepts. Therefore, the obtained age values of the syn-kinematic minerals are interpreted as formation ages which date increments of a long lasting deformation period. The time range of deformation of each shear zone system is bracketed by the oldest and youngest formation ages of syn-kinematic phengite and K-feldspar. Post-kinematic phengite laths show the youngest formation ages and overlap with the youngest syn-kinematic formation ages. This relationship indicates that post-kinematic growth occurred immediately after syn-kinematic mineral formation at the end of ductile sinistral shear. Hence, the

  10. In-Situ Chemical Analyses of Mineral Inclusions in Diamonds in Kimberlitic Eclogites From Yakutia

    NASA Astrophysics Data System (ADS)

    ANAND, M.; MISRA, K. C.; TAYLOR, L. A.; SOBOLEV, N. V.

    2001-12-01

    Mineral inclusions in diamonds (DIs) are stated to provide P-T-X-t information regarding the formation of the diamonds and the nature of the upper mantle. In an endeavor to further understand the importance of diamonds and their DIs in relation to their host rocks, we have investigated several diamondiferous eclogites from Yakutia, first by HRXC tomography (Taylor et al., 2001, this meeting) and then by dissection of the eclogites into their individual minerals. The mineralogy of the host eclogite is presented by Misra et al. (2001, this meeting). Two of the diamondiferous eclogite xenoliths, although weighing but 66 g and 42 g, contain 74 and 47 macro-diamonds, resp. Based on HRXCT imaging, appropriate sections were selected in the eclogite to extract diamonds with minimum loss of material. In the majority of cases, diamonds occur as perfect octahedron with well developed crystal faces. In some cases, however, diamonds occur as macles (twinned xls). The size range of the diamonds is 1-6 mm. Optical examination reveals the sulfides as the most common DIs in these diamonds, followed by clinopyroxenes and garnets. Each diamond was cut and polished along relatively soft directions parallel to either (001) or (110) faces so as to expose DIs for in-situ analyses. Examination by cathodoluminescence (CL) on an EMP demonstrated that the majority of the diamonds have minute, optically invisible, cracks from the DIs to the surfaces of the diamonds - i.e., the possibility of an open system. These diamonds show complicated growth histories and contain DIs that are in some cases, found to be associated with secondary alteration. In addition, the DIs in each diamond, examined in-situ are of different composition from the host and different from DIs in other diamonds, a relationship reported earlier (Taylor et al., 2000, Int'l Geol Rev). These observations raise serious doubts about the significance of DIs and the pristinity and syngenesis of DIs removed by the typical diamond

  11. Homoeologous chromosome pairing in the distant hybrid Alstroemeria aurea x A. inodora and the genome composition of its backcross derivatives determined by fluorescence in situ hybridization with species-specific probes.

    PubMed

    Kamstra, S A; Ramanna, M S; de Jeu, M J; Kuipers, A G; Jacobsen, E

    1999-01-01

    A distant hybrid between two diploid species (2n = 2x = 16), Alstroemeria aurea and A. inodora, was investigated for homoeologous chromosome pairing, crossability with A. inodora and chromosome transmission to its BC1 offspring. Fluorescence in situ hybridization (FISH) with two species-specific probes, A001-I (A. aurea specific) and D32-13 (A. inodora specific), was used to analyse chromosome pairing in the hybrid and the genome constitution of its BC1 progeny plants. High frequencies of associated chromosomes were observed in both genotypes of the F1 hybrid, A1P2-2 and A1P4. In the former, both univalents and bivalents were found at metaphase I, whereas the latter plant also showed tri- and quadrivalents. Based on the hybridization sites of DNA probes on the chromosomes of both parental species, it was established that hybrid A1P4 contains a reciprocal translocation between the short arm of chromosome 1 and the long arm of chromosome 8 of A. inodora. Despite regular homoeologous chromosome pairing in 30% of the pollen mother cells, both hybrids were highly sterile. They were backcrossed reciprocally with one of the parental species, A. inodora. Two days after pollination, embryo rescue was applied and, eventually, six BC1 progeny plants were obtained. Among these, two were aneuploids (2n = 2x + 1 = 17) and four were triploids (2n = 3x = 24). The aneuploid plants had originated when the interspecific hybrid was used as a female parent, indicating that n eggs were functional in the hybrid. In addition, 2n gametes were also functional in the hybrid, resulting in the four triploid BC1 plants. Of these four plants, three had received 2n pollen grains from the hybrid and one a 2n egg. Using FISH, homoeologous crossing over between the chromosomes of the two parental species in the hybrid was clearly detected in all BC1 plants. The relevance of these results for the process of introgression and the origin of n and 2n gametes are discussed. PMID:10087627

  12. Evidence for in-situ methane production in ice based on anomalous isotope analyses

    NASA Astrophysics Data System (ADS)

    Sowers, T. A.; Priscu, J.

    2004-12-01

    the Sajama ice core from central Bolivia (18oS, 69oW, 6542masl), for example, were 1X-5X higher than contemporaneous values recorded in polar ice cores [Campen et al., 2003]. \\delta13CH4 values from five discrete depths were compared to corresponding measurements made on the Taylor Dome ice core and suggest the additional (in-situ) CH_{4} in the Sajama samples has an average isotopic composition of -63.2±2.8‰ . For reference, atmospheric δ ^{13}CH_{4} values range from -42 to -45/pm over this period. The Sajama isotope values are characteristic of methanogenic CH_{4} emitted from most terrestrial ecosystems. The second case study revolves around ice that was recovered from a perennially ice covered lake in the McMurdo Dry Valleys, Antarctica. Previous work on ice from Lake Bonney demonstrated a rich microbial consortium located ~2m below the surface [Priscu et al., 1998]. Methane isotope analyses were made on ice from this depth interval to identify the presence of microbially produced CH_{4}. δ ^{13}CH_{4} and δ DCH4 results suggest the CH4 arises from acetogenic CH4 production as opposed to CO2 reduction. Campen, R.K., T. Sowers, and R.B. Alley, Evidence of Microbial Consortia Metabolizing Within a Low-Latitude Mountain Glacier, Geology, 31 (No. 3), 231-234, 2003. Priscu, J.C., et al., Perennial Antarctic Lake Ice: An oasis for life in a polar desert, Science, 280, 2095-2098, 1998.

  13. In situ hybridization assay-based small molecule screening in zebrafish

    PubMed Central

    Jing, Lili; Durand, Ellen M.; Ezzio, Catherine; Pagliuca, Stephanie M.; Zon, Leonard I.

    2012-01-01

    In vitro biochemical and cell-based small molecule screens have been widely used to identify compounds that target specific signaling pathways. But the identified compounds frequently fail at the animal testing stage, largely due to the in vivo absorption, metabolism and toxicity of chemicals. Zebrafish has recently emerged as a vertebrate whole organism model for small molecule screening. The in vivo bioactivity and specificity of compounds are examined from the very beginning of zebrafish screens. In addition, zebrafish is suitable for chemical screens at a large scale similar to cellular assays. This protocol describes an approach for in situ hybridization (ISH)-based chemical screening in zebrafish, which, in principle, can be used to screen any gene product. The described protocol has been used to identify small molecules affecting specific molecular pathways and biological processes. It can also be adapted to zebrafish screens with different readouts. PMID:23001521

  14. Fluorescence in situ hybridization (FISH) mapping of single copy genes on Trichomonas vaginalis chromosomes.

    PubMed

    Zubáčová, Zuzana; Krylov, Vladimír; Tachezy, Jan

    2011-04-01

    The highly repetitive nature of the Trichomonas vaginalis genome and massive expansion of various gene families has caused difficulties in genome assembly and has hampered genome mapping. Here, we adapted fluorescence in situ hybridization (FISH) for T. vaginalis, which is sensitive enough to detect single copy genes on metaphase chromosomes. Sensitivity of conventional FISH, which did not allow single copy gene detection in T. vaginalis, was increased by means of tyramide signal amplification. Two selected single copy genes, coding for serine palmitoyltransferase and tryptophanase, were mapped to chromosome I and II, respectively, and thus could be used as chromosome markers. This established protocol provides an amenable tool for the physical mapping of the T. vaginalis genome and other essential applications, such as development of genetic markers for T. vaginalis genotyping. PMID:21195113

  15. HIV detection by in-situ hybridization based on confocal reflected light microscopy

    NASA Astrophysics Data System (ADS)

    Smith, Louis C.; Jericevic, Zeljko; Cuellar, Roland; Paddock, Stephen W.; Lewis, Dorothy E.

    1991-05-01

    Elucidation of the pathogenesis of AIDS is confounded by the finding that few actively infected CD4+ cells (1 in 104-105) can be detected in the peripheral blood, even though there is dramatic depletion (often >90%) of CD4+ cells as the disease progresses. A sensitive, 35S-based human immunodeficiency virus (HIV) mRNA in situ hybridization technique was coupled with a new detection method, confocal laser scanning microscopy, to examine transcriptionally active HIV-infected cells from individuals at different disease stages. An algorithm for image segmentation and analysis has been developed to determine the proportion of HIV-positive cells. Data obtained using this improved detection method suggest that there are more HIV mRNA-producing cells in HIV-infected individuals than previously thought, based on other detection methods.

  16. Development of single-cell array for large-scale DNA fluorescence in situ hybridization

    PubMed Central

    Liu, Yingru; Kirkland, Brett; Shirley, James; Wang, Zhibin; Zhang, Peipei; Stembridge, Jacquelyn; Wong, Wilson; Takebayashi, Shin-ichiro; Gilbert, David M.; Lenhert, Steven

    2013-01-01

    DNA fluorescence in situ hybridization (FISH) is a powerful cytogenetic assay, but conventional sample-preparation methods for FISH do not support large-scale high-throughput data acquisition and analysis, which are potentially useful for several biomedical applications. To address this limitation, we have developed a novel FISH sample-preparation method based on generating a centimetre-sized cell array, in which all cells are precisely positioned and separated from their neighbours. This method is simple and easy and capable of patterning nonadherent human cells. We have successfully performed DNA FISH on the single-cell arrays, which facilitate analysis of FISH results with the FISH-FINDER computer program. PMID:23370691

  17. E-cadherin expression in colorectal cancer. An immunocytochemical and in situ hybridization study.

    PubMed Central

    Dorudi, S.; Sheffield, J. P.; Poulsom, R.; Northover, J. M.; Hart, I. R.

    1993-01-01

    Expression of the epithelial-specific adhesion molecule E-cadherin has been assessed in paraffin-embedded tissue from a series of 72 colorectal carcinomas. Using immunocytochemistry and in situ hybridization it was found that E-cadherin expression was related inversely to tumor differentiation. Out of 44 well- and moderately differentiated tumors, 36 expressed good positivity, whereas 24 of 28 poorly differentiated tumors were E-cadherin-negative. Classification by Dukes stage revealed a highly significant difference (P << 0.001) between A and B (32 positive, four negative) and C1 and C2 (seven positive, 29 negative) stages in terms of immunoreactivity. Of the 32 lymph node metastases studied, 20 were negative for E-cadherin expression, as were seven of eight liver metastases. These results indicate that the down-regulation of E-cadherin levels in vivo is associated with the dedifferentiation, progression, and metastasis of colorectal cancer. Images Figure 1 Figure 2 PMID:7682766

  18. Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)

    SciTech Connect

    Deutsch, Eric W.; Ball, Cathy; Berman, Jules J.; Bova, G. S.; Brazma, Alvis; Bumgarner, Roger E.; Campbell, David; Causton, Helen C.; Christiansen, Jeff; Daian, Fabrice; Dauga, Delphine; Davidson, Duncan; Gimenez, Gregory; Goo, Young Ah; Grimmond, Sean; Henrich, Thorsten; Herrmann, Bernhard G.; Johnson, Michael H.; Korb, Martin; Mills, Jason C.; Oudes, Asa; Parkinson, Helen E.; Pascal, Laura E.; Pollet, Nicolas; Quackenbush, John; Ramialison, Mirana; Ringwald, Martin; Salgado, David; Sansone, Susanna A.; Sherlock, Gavin; Stoeckert, Christian Jr. J.; Swedlow, Jason; Taylor, Ronald C.; Walashek, Laura; Warford, Anthony; Wilkinson, David G.; Zhou, Yi; Zon, Leonard I.; Liu, Alvin Y.; True, Lawrence D.

    2008-03-03

    We describe the creation process of the Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE). Modeled after the existing minimum information specification for microarray data, we created a new specification for gene expression localization experiments, initially to facilitate data sharing within a consortium. After successful use within the consortium, the specification was circulated to members of the wider biomedical research community for comment and refinement. After a period of acquiring many new suggested requirements, it was necessary to enter a final phase of excluding those requirements that were deemed inappropriate as a minimum requirement for all experiments. The full specification will soon be published as a version 1.0 proposal to the community, upon which a more full discussion must take place so that the final specification may be achieved with the involvement of the whole community.

  19. Chromosome translocations measured by fluorescence in-situ hybridization: A promising biomarker

    SciTech Connect

    Lucas, J.N.; Straume, T.

    1995-10-01

    A biomarker for exposure and risk assessment would be most useful if it employs an endpoint that is highly quantitative, is stable with time, and is relevant to human risk. Recent advances in chromosome staining using fluorescence in situ hybridization (FISH) facilitate fast and reliable measurement of reciprocal translocations, a kind of DNA damage linked to both prior exposure and risk. In contrast to other biomarkers available, the frequency of reciprocal translocations in individuals exposed to whole-body radiation is stable with time post exposure, has a rather small inter-individual variability, and can be measured accurately at the low levels. Here, the authors discuss results from their studies demonstrating that chromosome painting can be used to reconstruct radiation dose for workers exposed within the dose limits, for individuals exposed a long time ago, and even for those who have been diagnosed with leukemia but not yet undergone therapy.

  20. Acquired cystic disease-associated renal cell carcinoma: an immunohistochemical and fluorescence in situ hybridization study.

    PubMed

    Kuroda, Naoto; Yamashita, Motoki; Kakehi, Yoshiyuki; Hes, Ondrej; Michal, Michal; Lee, Gang-Hong

    2011-12-01

    Acquired cystic disease (ACD)-associated renal cell carcinoma (RCC) has been recently identified. However, there are only a few genetic studies to date. In this article, we performed an immunohistochemical and fluorescence in situ hybridization (FISH) study for six cases including one case with sarcomatoid change. As a result, we observed frequent immunohistochemical expression of AMACR. FISH of chromosome 3 showed trisomy for three cases, monosomy for two cases, and disomy for one case. Additionally, FISH of chromosome 16 showed trisomy for three cases, monosomy for two cases, and both trisomy and monosomy for one case. Furthermore, both the carcinomatous area and the sarcomatoid area of one ACD-associated RCC with sarcomatoid change revealed monosomy of chromosomes 3, 9, and 16 but showed disomy of chromosome 14. In conclusion, the numerical abnormalities of chromosomes 3 and 16, irrespective of gain or loss, may be characteristic of ACD-associated RCC. PMID:22179186

  1. Identification of supernumerary ring chromosome 1 mosaicism using fluorescence in situ hybridization

    SciTech Connect

    Chen, H.; Tuck-Muller, C.M.; Wertelecki, W.

    1995-03-27

    We report on a 15-year-old black boy with severe mental retardation, multiple congenital anomalies, and a supernumerary ring chromosome mosaicism. Fluorescence in situ hybridization with a chromosome 1 painting probe (pBS1) identified the ring as derived from chromosome 1. The karyotype was 46,XY/47,XY,+r(1)(p13q23). A review showed 8 reports of ring chromosome 1. In 5 cases, the patients had a non-supernumerary ring chromosome 1 resulting in partial monosomies of the short and/or long arm of chromosome 1. In 3 cases, the presence of a supernumerary ring resulted in partial trisomy of different segments of chromosome 1. In one of these cases of the supernumerary ring was composed primarily of the centromere and the heterochromatic region of chromosome 1, resulting in normal phenotype. Our patient represents the third report of a supernumerary ring chromosome 1 resulting in abnormal phenotype. 28 refs., 5 figs., 1 tab.

  2. Partial trisomy 13q identified by sequential fluorescence in situ hybridization

    SciTech Connect

    Gopal Rao, V.V.N.; Carpenter, N.J.; Gucsavas, M.

    1995-07-31

    We report on a 19-month-old boy with partial trisomy 13q resulting from a probable balanced translocation involving chromosomes 1 and 13. The infant presented with omphalocele, malrotation, microcephaly with overriding skull bones, micrognathia, apparently low-set ears, rocker-bottom feet, and congenital heart disease, findings suggestive of trisomy 13. Karyotypic studies from peripheral blood lymphocytes documented an unbalanced karyotype 46,XY,-1,+der. The mother`s chromosomes were normal, and the father was not available. Conventional cytogenetic techniques were unable to identify the extra material on the terminal 1q. Using fluorescence in situ hybridization (FISH) on the GTL-banded metaphases, the extra material on 1q was identified as the terminal long arm of 13, thus resulting in partial trisomy 13 (q32-qter). 8 refs., 2 figs., 1 tab.

  3. Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH).

    PubMed

    Lecrenier, M C; Ledoux, Q; Berben, G; Fumière, O; Saegerman, C; Baeten, V; Veys, P

    2014-01-01

    Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as "mad cow disease"), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application. PMID:25034259

  4. Enumeration of methanogens with a focus on fluorescence in situ hybridization

    NASA Astrophysics Data System (ADS)

    Kumar, Sanjay; Dagar, Sumit Singh; Mohanty, Ashok Kumar; Sirohi, Sunil Kumar; Puniya, Monica; Kuhad, Ramesh C.; Sangu, K. P. S.; Griffith, Gareth Wyn; Puniya, Anil Kumar

    2011-06-01

    Methanogens, the members of domain Archaea are potent contributors in global warming. Being confined to the strict anaerobic environment, their direct cultivation as pure culture is quite difficult. Therefore, a range of culture-independent methods have been developed to investigate their numbers, substrate uptake patterns, and identification in complex microbial communities. Unlike other approaches, fluorescence in situ hybridization (FISH) is not only used for faster quantification and accurate identification but also to reveal the physiological properties and spatiotemporal dynamics of methanogens in their natural environment. Aside from the methodological aspects and application of FISH, this review also focuses on culture-dependent and -independent techniques employed in enumerating methanogens along with associated problems. In addition, the combination of FISH with micro-autoradiography that could also be an important tool in investigating the activities of methanogens is also discussed.

  5. Chromosomal loci of 50 human keratinocyte cDNAs assigned by fluorescence in situ hybridization

    SciTech Connect

    Morishima, Yohich; Ariyama, Takeshi; Yamanishi, Kiyofumi

    1995-07-20

    The chromosomal loci of expressed genes provide useful information for a candidate gene approach to the genes responsible for genetic diseases. A large set of randomly isolated cDNAs catalogued by partial sequencing can serve as a resource for accessing and isolating these disease genes. Using fluorescence in situ hybridization, we examined the chromosomal loci of 217 human keratinocyte-derived cDNAs, with independent novel sequence tags at the 3{prime} end region. Among them, we determined the loci of 50 cDNAs. Single-pass sequencing of these from the 5{prime} ends indicated that 39 cDNAs still can be produced for new genes. These cDNAs with identified chromosomal loci are powerful tools that can be used to help elucidate the genes responsible for hereditary skin disorders. 42 refs., 3 figs., 2 tabs.

  6. Blue Nevus-Like Metastasis of a Cutaneous Melanoma Identified by Fluorescence In Situ Hybridization.

    PubMed

    Campa, Molly; Patel, Mahir; Aubert, Pamela; Hosler, Gregory; Witheiler, Daniel

    2016-09-01

    A blue nevus-like melanoma is a rare melanoma variant arising from or histologically similar to a blue nevus. It can be challenging to distinguish a cellular blue nevus from a blue nevus-like melanoma, particularly in cases of blue nevus-like melanoma lacking a transition from a clearly benign component. We present a case of a 78-year-old man who refused treatment for a previously existing melanoma and subsequently developed a gray nodule near the site of the previous melanoma. After fluorescence in situ hybridization revealed copy number gains in RREB1, this was diagnosed as a blue nevus-like metastatic melanoma. Blue nevus-like metastatic melanoma is most commonly seen near the site of the primary cutaneous melanoma. This entity should be considered in a patient with a history of melanoma and a new blue nevus-like lesion. PMID:27097332

  7. Chromosome-Specific DNA Repeats: Rapid Identification in Silico and Validation Using Fluorescence in Situ Hybridization

    PubMed Central

    Hsu, Joanne H.; Zeng, Hui; Lemke, Kalistyn H.; Polyzos, Aris A.; Weier, Jingly F.; Wang, Mei; Lawin-O’Brien, Anna R.; Weier, Heinz-Ulrich G.; O’Brien, Benjamin

    2013-01-01

    Chromosome enumeration in interphase and metaphase cells using fluorescence in situ hybridization (FISH) is an established procedure for the rapid and accurate cytogenetic analysis of cell nuclei and polar bodies, the unambiguous gender determination, as well as the definition of tumor-specific signatures. Present bottlenecks in the procedure are a limited number of commercial, non-isotopically labeled probes that can be combined in multiplex FISH assays and the relatively high price and effort to develop additional probes. We describe a streamlined approach for rapid probe definition, synthesis and validation, which is based on the analysis of publicly available DNA sequence information, also known as “database mining”. Examples of probe preparation for the human gonosomes and chromosome 16 as a selected autosome outline the probe selection strategy, define a timeline for expedited probe production and compare this novel selection strategy to more conventional probe cloning protocols. PMID:23344021

  8. Fluorescent in situ hybridization (FISH) assessment of chromosome copy number in sperm

    SciTech Connect

    Sheu, M.; Sigman, M.; Mark, H.F.L.

    1994-09-01

    Approximately 15% of all recognized pregnancies end in spontaneous abortions. The overall frequency of chromosome abnormalities in spontaneous abortions is approximately 50%. Thus aneuploidy is a significant cause of fetal wastage. In addition, structural and numerical abnormalities of chromosomes can also lead to birth defects, developmental delay, mental retardation and infertility. Conventional cytogenetic analysis via GTG- and other banding techniques is a powerful tool in the elucidation of the nature of chromosomal abnormalities. Fluorescent in situ hybridization (FISH) enables detection of numerical chromosomal abnormalities, especially trisomies, in intact cells. Using FISH and commercially available biotin-labeled probes, we have initiated a prospective study to assess specific chromosome copy number of preparations of unstained smears from men referred for a male infertility evaluation as well as smears from normal control males chosen randomly from the sample of sperm donors. A total of approximately 19,000 sperm nuclei have been examined thus far. Of those suitable for analysis, 7382 (38.75%) were normal possessing one copy of chromosome 8, 155 (0.81%) were disomic, and 15 (0.079%) had more than two copies of chromosome 8. Comparisons with data available in the literature will be discussed. Work is ongoing to increase the efficiency of hybridization using both reported and previously untried pretreatment and fixation protocols. We have also initiated studies using multicolor FISH with various chromosome enumeration probes. The assay described here is a potentially powerful tool for detecting rare events such as spontaneous germ cell aneuploidy, aneuploidy detected in semen from men with carcinoma in situ of the testis and aneuploidy induced by potential environmental genotoxicants. It can also be utilized for segregation analysis and for correlating chromosome copy number with germ cell morphology.

  9. Fluorescence in-situ hybridization (FISH) as a tool for visualization and enumeration of Campylobacter in broiler ceca

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Food-borne human pathogens are typically detected and enumerated by either cultural methods or PCR-based approaches. Fluorescence in-situ hybridization (FISH) is a standard microscopy tool for microbial ecology but has not been widely used for food safety applications despite important advantages o...

  10. RAPID IDENTIFICATION OF CANDIDA ALBICANS DIRECTLY FROM YEAST POSITIVE BLOOD CULTURE BOTTLES BY FLUORESCENCE IN SITU HYBRIDIZATION USING PNA PROBES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for identification of Candida albicans directly from yeast-positive blood culture bottles is described. The test (C. albicans PNA FISH) is based on a fluorescein-labeled PNA probe targeting C. albicans 26...

  11. Fluorescence in situ hybridization for rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis recovered from cystic fibrosis patients.

    PubMed

    Wellinghausen, Nele; Wirths, Beate; Poppert, Sven

    2006-09-01

    Achromobacter xylosoxidans is frequently isolated from the respiratory secretions of cystic fibrosis (CF) patients, but identification with biochemical tests is unreliable. We describe fluorescence in situ hybridization assays for the rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis. Both assays showed high sensitivities and high specificities with a collection of 155 nonfermenters from CF patients. PMID:16954289

  12. Genomic in situ hybridization identifies parental chromosomes in hybrid scallop (Bivalvia, Pectinoida, Pectinidae) between female Chlamys farreri and male Argopecten irradians irradians

    PubMed Central

    Huang, Xiaoting; Bi, Ke; Lu, Wei; Wang, Shi; Zhang, Lingling; Bao, Zhenmin

    2015-01-01

    Abstract Interspecific crossing was artificially carried out between Chlamys farreri (Jones & Preston, 1904) ♀ and Argopecten irradians irradians (Lamarck, 1819) ♂, two of the dominant cultivated scallop species in China. Genomic in situ hybridization (GISH) was used to examine the chromosome constitution and variation in hybrids at early embryonic stage. The number of chromosomes in 66.38% of the metaphases was 2n = 35 and the karyotype was 2n = 3 m + 5 sm + 16 st + 11 t. After GISH, two parental genomes were clearly distinguished in hybrids, most of which comprised 19 chromosomes derived from their female parent (Chlamys farreri) and 16 chromosomes from their male parent (Argopecten irradians irradians). Some chromosome elimination and fragmentation was also observed in the hybrids. PMID:26140161

  13. Genetic evidence of hybridization between the critically endangered Cuban crocodile and the American crocodile: implications for population history and in situ/ex situ conservation

    PubMed Central

    Milián-García, Y; Ramos-Targarona, R; Pérez-Fleitas, E; Sosa-Rodríguez, G; Guerra-Manchena, L; Alonso-Tabet, M; Espinosa-López, G; Russello, M A

    2015-01-01

    Inter-specific hybridization may be especially detrimental when one species is extremely rare and the other is abundant owing to the potential for genetic swamping. The Cuban crocodile (Crocodylus rhombifer) is a critically endangered island endemic largely restricted to Zapata Swamp, where it is sympatric with the widespread American crocodile (C. acutus). An on-island, C. rhombifer captive breeding program is underway with the goals of maintaining taxonomic integrity and providing a source of individuals for reintroduction, but its conservation value is limited by lack of genetic information. Here we collected mtDNA haplotypic and nuclear genotypic data from wild and captive C. rhombifer and C. acutus in Cuba to: (1) investigate the degree of inter-specific hybridization in natural (in situ) and captive (ex situ) populations; (2) quantify the extent, distribution and in situ representation of genetic variation ex situ; and (3) reconstruct founder relatedness to inform management. We found high levels of hybridization in the wild (49.1%) and captivity (16.1%), and additional evidence for a cryptic lineage of C. acutus in the Antilles. We detected marginally higher observed heterozygosity and allelic diversity ex situ relative to the wild population, with captive C. rhombifer exhibiting over twice the frequency of private alleles. Although mean relatedness was high in captivity, we identified 37 genetically important individuals that possessed individual mean kinship (MK) values lower than the population MK. Overall, these results will guide long-term conservation management of Cuban crocodiles for maintaining the genetic integrity and viability of this species of high global conservation value. PMID:25335559

  14. Genetic evidence of hybridization between the critically endangered Cuban crocodile and the American crocodile: implications for population history and in situ/ex situ conservation.

    PubMed

    Milián-García, Y; Ramos-Targarona, R; Pérez-Fleitas, E; Sosa-Rodríguez, G; Guerra-Manchena, L; Alonso-Tabet, M; Espinosa-López, G; Russello, M A

    2015-03-01

    Inter-specific hybridization may be especially detrimental when one species is extremely rare and the other is abundant owing to the potential for genetic swamping. The Cuban crocodile (Crocodylus rhombifer) is a critically endangered island endemic largely restricted to Zapata Swamp, where it is sympatric with the widespread American crocodile (C. acutus). An on-island, C. rhombifer captive breeding program is underway with the goals of maintaining taxonomic integrity and providing a source of individuals for reintroduction, but its conservation value is limited by lack of genetic information. Here we collected mtDNA haplotypic and nuclear genotypic data from wild and captive C. rhombifer and C. acutus in Cuba to: (1) investigate the degree of inter-specific hybridization in natural (in situ) and captive (ex situ) populations; (2) quantify the extent, distribution and in situ representation of genetic variation ex situ; and (3) reconstruct founder relatedness to inform management. We found high levels of hybridization in the wild (49.1%) and captivity (16.1%), and additional evidence for a cryptic lineage of C. acutus in the Antilles. We detected marginally higher observed heterozygosity and allelic diversity ex situ relative to the wild population, with captive C. rhombifer exhibiting over twice the frequency of private alleles. Although mean relatedness was high in captivity, we identified 37 genetically important individuals that possessed individual mean kinship (MK) values lower than the population MK. Overall, these results will guide long-term conservation management of Cuban crocodiles for maintaining the genetic integrity and viability of this species of high global conservation value. PMID:25335559

  15. Development of a fluorescent in situ hybridization (FISH) technique for visualizing CGMMV in plant tissues.

    PubMed

    Shargil, D; Zemach, H; Belausov, E; Lachman, O; Kamenetsky, R; Dombrovsky, A

    2015-10-01

    Cucumber green mottle mosaic virus (CGMMV), which belongs to the genus Tobamovirus, is a major pathogen of cucurbit crops grown indoors and in open fields. Currently, immunology (e.g., ELISA) and molecular amplification techniques (e.g., RT-PCR) are employed extensively for virus detection in plant tissues and commercial seed lots diagnostics. In this study, a fluorescent in situ hybridization (FISH) technique, using oligonucleotides whose 5'-terminals were labeled with red cyanine 3 (Cy3) or green fluorescein isothiocyanate (FITC), was developed for the visualization of the pathogen in situ. This simple and reliable method allows detection and localization of CGMMV in the vegetative and reproductive tissues of cucumber and melon. When this technique was applied in male flowers, anther tissues were found to be infected; whereas the pollen grains were found to be virus-free. These results have meaningful epidemiological implications for the management of CGMMV, particularly with regard to virus transfer via seed and the role of insects as CGMMV vectors. PMID:26231788

  16. Fluorescence in situ hybridization for the identification of Treponema pallidum in tissue sections.

    PubMed

    Petrich, Annett; Rojas, Pablo; Schulze, Julia; Loddenkemper, Christoph; Giacani, Lorenzo; Schneider, Thomas; Hertel, Moritz; Kikhney, Judith; Moter, Annette

    2015-10-01

    Syphilis is often called the great imitator because of its frequent atypical clinical manifestations that make the disease difficult to recognize. Because Treponema pallidum subsp. pallidum, the infectious agent of syphilis, is yet uncultivated in vitro, diagnosis is usually made using serology; however, in cases where serology is inconclusive or in patients with immunosuppression where these tests may be difficult to interpret, the availability of a molecular tool for direct diagnosis may be of pivotal importance. Here we present a fluorescence in situ hybridization (FISH) assay that simultaneously identifies and analyzes spatial distribution of T. pallidum in histological tissue sections. For this assay the species-specific FISH probe TPALL targeting the 16S rRNA of T. pallidum was designed in silico and evaluated using T. pallidum infected rabbit testicular tissue and a panel of non-syphilis spirochetes as positive and negative controls, respectively, before application to samples from four syphilis-patients. In a HIV positive patient, FISH showed the presence of T. pallidum in inguinal lymph node tissue. In a patient not suspected to suffer from syphilis but underwent surgery for phimosis, numerous T. pallidum cells were found in preputial tissue. In two cases with oral involvement, FISH was able to differentiate T. pallidum from oral treponemes and showed infection of the oral mucosa and tonsils, respectively. The TPALL FISH probe is now readily available for in situ identification of T. pallidum in selected clinical samples as well as T. pallidum research applications and animal models. PMID:26365167

  17. High-resolution telomere fluorescence in situ hybridization reveals intriguing anomalies in germ cell tumors.

    PubMed

    Shekhani, Mohammed Talha; Barber, John R; Bezerra, Stephania M; Heaphy, Christopher M; Gonzalez Roibon, Nilda Diana; Taheri, Diana; Reis, Leonardo O; Guner, Gunes; Joshu, Corinne E; Netto, George J; Meeker, Alan K

    2016-08-01

    Testicular germ cell tumor (TGCT) is the most common malignancy of young men. Most patients are completely cured, which distinguishes these from most other malignancies. Orchiectomy specimens (n=76) were evaluated using high-resolution (single-cell discriminative) telomere-specific fluorescence in situ hybridization (FISH) with simultaneous Oct4 immunofluorescence to describe telomere length phenotype in TGCT neoplastic cells. For the first time, the TGCT precursor lesion, germ cell neoplasia in situ (GCNIS) is also evaluated in depth. The intensity of the signals from cancerous cells was compared to the same patient's reference cells-namely, healthy germ cells (defined as "medium" length) and interstitial/somatic cells (defined as "short" telomere length). We observed short telomeres in most GCNIS and pure seminomas (P=.006 and P=.0005, respectively). In contrast, nonseminomas displayed longer telomeres. Lesion-specific telomere lengths were documented in mixed tumor cases. Embryonal carcinoma (EC) demonstrated the longest telomeres. A fraction of EC displays the telomerase-independent alternative lengthening of telomeres (ALT) phenotype (24% of cases). Loss of ATRX or DAXX nuclear expression was strongly associated with ALT; however, nuclear expression of both proteins was retained in half of ALT-positive ECs. The particular distribution of telomere lengths among TGCT and GCNIS precursors implicate telomeres anomalies in pathogenesis. These results may advise management decisions as well. PMID:27085557

  18. Simultaneous specific in planta visualization of root-colonizing fungi using fluorescence in situ hybridization (FISH).

    PubMed

    Vági, Pál; Knapp, Dániel G; Kósa, Annamária; Seress, Diána; Horváth, Áron N; Kovács, Gábor M

    2014-05-01

    In planta detection of mutualistic, endophytic, and pathogenic fungi commonly colonizing roots and other plant organs is not a routine task. We aimed to use fluorescence in situ hybridization (FISH) for simultaneous specific detection of different fungi colonizing the same tissue. We have adapted ribosomal RNA (rRNA) FISH for visualization of common mycorrhizal (arbuscular- and ectomycorrhiza) and endophytic fungi within roots of different plant species. Beside general probes, we designed and used specific ones hybridizing to the large subunit of rRNA with fluorescent dyes chosen to avoid or reduce the interference with the autofluorescence of plant tissues. We report here an optimized efficient protocol of rRNA FISH and the use of both epifluorescence and confocal laser scanning microscopy for simultaneous specific differential detection of those fungi colonizing the same root. The method could be applied for the characterization of other plant-fungal interactions, too. In planta FISH with specific probes labeled with appropriate fluorescent dyes could be used not only in basic research but to detect plant colonizing pathogenic fungi in their latent life-period. PMID:24221902

  19. Postembedding ultrastructural in situ hybridization on ultrathin cryosections and LR white resin sections.

    PubMed

    Mandry, P; Murray, B A; Rieke, L; Becke, H; Höfler, H

    1993-01-01

    A method was developed for nonisotopic postembedding in situ hybridization (ISH) on ultrathin sections of frozen and of LR White resin-embedded material at the electron microscopic level. The method was successfully applied to detect Epstein-Barr virus (EBV) DNA in the P3HR1 human Burkitt's lymphoma cell line. Each of the steps in the procedure had to be optimized for successful ISH on the frozen and LR White sections. The most important conditions are described. Predigestion with proteinase K was only necessary with the resin sections. Sections were treated with sodium hydroxide to denature target DNA and were hybridized with a biotinylated probe. The probe was best detected with a primary antibody to biotin followed by a gold-conjugated secondary antibody. EBV DNA was detected in the nucleus and/or cytoplasm in 10% to 20% of P3HR1 cells. A similar percentage of cells in thin L-sectioned material prepared by routine methods showed virus particles at different stages of maturation. PMID:8391176

  20. X chromosome aneuploidy in infertile women: Analysis by interphase fluorescent in situ hybridization

    SciTech Connect

    Morris, M.A.; Moix, I.; Mermillod, B.

    1994-09-01

    Up to 1 in 3 couples have a problem of infertility at some time in their lives. Sex chromosome anomalies are found in 5-10% of couples, with mosaic aneuploidy being a common finding in primary infertility. Recurrent spontaneous abortion (RSA), in contrast, is frequently associated with autosomal structural anomalies. We hypothesized that low-level mosaic X chromosome aneuploidy was associated with primary infertility but not with RSA. Three groups were studied: women from couples with primary infertillity (n=26); women with three or more spontaneous abortions (n=22); and age-matched normally fertile women (at least two pregnancies; n=28). Interphase fluorescent in situ hybridization (FISH) was used to determine X chromosome ploidy in 100 nuclei per patient, using a contig of three cosmids from MAO locus (kindly donated by W. Berger, Nijmegen). A control probe (chr. 15 centromere) was simultaneously hybridized, and only nuclei containing two control signals were scored for the X chromosome. The mean numbers of nuclei with two X chromosome signals were the same in all groups (Welch equality of means test: p>0.97). However, there is a significant difference between the variances of the primary infertile and RSA groups (Levene`s test: p=0.025 after Bonferrone correction for multiple testing). This provides preliminary support for the hypothesis of an association between primary infertility and low-level mosaic X chromosome aneuploidy.

  1. Intracellular location of rabbit poxvirus nucleic acid within infected cells as determined by in situ hybridization.

    PubMed Central

    Minnigan, H; Moyer, R W

    1985-01-01

    The intracellular location of rabbit poxvirus DNA within cells during the course of infection has been determined by the hybridization in situ of labeled viral DNA probes to uninfected and infected cells under various conditions. Extensive control experiments were performed to demonstrate that DNA could be detected selectively and accurately within the cell. Our results suggest that rabbit poxvirus DNA is located only within the cytoplasm during the reproductive cycle, and we found no evidence that viral DNA enters the cell nucleus. The pattern of hybridization of viral DNA at early times (1 and 2 h postinfection) and in the presence of inhibitors of viral DNA synthesis suggests that there may be an association between the input viral DNA and some structural component of the host cell. A number of observations support the hypothesis that the host cell nucleus is required for a productive poxvirus infection. Our results are discussed in terms of the possible role of the nucleus in the replication of poxviruses. Images PMID:2991586

  2. Calibration of interphase fluorescence in situ hybridization cutoff by mathematical models.

    PubMed

    Du, Qinghua; Li, Qingshan; Sun, Daochun; Chen, Xiaoyan; Yu, Bizhen; Ying, Yi

    2016-03-01

    Fluorescence in situ hybridization (FISH) continues to play an important role in clinical investigations. Laboratories may create their own cutoff, a percentage of positive nuclei to determine whether a specimen is positive or negative, to eliminate false positives that are created by signal overlap in most cases. In some cases, it is difficult to determine the cutoff value because of differences in both the area of nuclei and the number of signals. To address these problems, we established two mathematical models using probability theory. To verify these two models, normal disomy cells from healthy individuals were used to simulate cells with different numbers of signals by hybridization with different probes. We used an X/Y probe to obtain the average distance between two signals and the probability of signal overlap in different nuclei area. Frequencies of all signal patterns were scored and compared with theoretical frequencies, and models were assessed using a goodness of fit test. We used five BCR/ABL1-positive samples, 20 BCR/ABL1-negative samples and two samples with ambiguous results to verify the cutoff calibrated by these two models. The models were in agreement with experimental results. The dynamic cutoff can classify cases in routine analysis correctly, and it can also correct for influences from nuclei area and the number of signals in some ambiguous cases. The probability models can be used to assess the effect of signal overlap and calibrate the cutoff. © 2015 International Society for Advancement of Cytometry. PMID:26580488

  3. Localization of the expression of complement component 3 in the human endometrium by in situ hybridization

    SciTech Connect

    Sayegh, R.A.; Tao, Xiao Jing; Awwad, J.T.

    1996-04-01

    C3 production by the human endometrium has been previously described. The objective of the current study was to localize the site of expression and regulation of the third component of complement, C3, in the endometrium. Eight secretory and eight proliferative archival endometrial samples from hysterectomy and endometrial biopsy specimens were used for in situ hybridization analysis. This analysis was performed with a radiolabeled riboprobe synthesized from a 736-bp template representing sequence 1944-2680 of the human C3 complementary DNA. Duplicate sections were hybridized with sense and antisense riboprobes. Resultant autoradiograms were analyzed qualitatively by light- and darkfield microscopy. In proliferative endometrium, minimal expression of C3 was observed and was limited to a few stromal patches and glands throughout the section. In the secretory samples, prominent C3 expression was observed in both the glands and stroma of the basalis layer. Endometrial lymphocytes did not express C3. Endometrial stromal and glandular cells express the C3 gene. Endometrial lymphocytes did not express C3, but other nondistinct lymphoid elements scattered in the stroma may be expressing C3. There was a visibly more intense expression of C3 in the basalis layer of the secretory endometrium than in proliferative endometrium. The spatial and temporal pattern of C3 expression may have implications in normal menstrual physiology and in the immunological response of the endometrium to the invading trophoblast during placentation. 23 refs., 4 figs., 1 tab.

  4. Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis.

    PubMed

    Milinovich, Gabriel J; Trott, Darren J; Burrell, Paul C; Croser, Emma L; Al Jassim, Rafat A M; Morton, John M; van Eps, Andrew W; Pollitt, Christopher C

    2007-08-01

    Carbohydrate-induced laminitis in horses is characterized by marked changes in the composition of the hindgut microbiota, from a predominantly Gram-negative population to one dominated by Gram-positive bacteria. The objective of this study was to monitor changes in the relative abundance of selected hindgut bacteria that have previously been implicated in the pathophysiology of equine laminitis using fluorescence in situ hybridization (FISH). Caecal cannulae were surgically implanted in five Standardbred horses and laminitis induced by oral administration of a bolus dose of oligofructose. Caecal fluid and faecal specimens were collected over a 48 h period at 2 to 4 h intervals post-oligofructose administration and subjected to FISH using probes specific for nine bacterial groups to determine changes in their relative abundance compared with total bacteria hybridizing to the generic EUBMIX probe. Additionally, hoof biopsies were taken over the course of the experiment at 6 h intervals and evaluated for histopathological changes consistent with laminitis, allowing changes in hindgut microbiota to be correlated with the onset of lesions in the foot. Of the microorganisms specifically targeted, streptococci of the Streptococcus bovis/equinus complex were the only bacteria that consistently proliferated in both caecal fluid and faeces immediately before the onset of histological signs of laminitis. Furthermore, lactobacilli, Enterobacteriaceae, Allisonella histaminiformans, enterococci, Bacteroides fragilis, Mitsuokella jalaludinii and Clostridium difficile did not establish significant populations in the hindgut before the onset of equine laminitis. PMID:17635552

  5. Single-mRNA counting using fluorescent in situ hybridization in budding yeast

    PubMed Central

    Trcek, Tatjana; Chao, Jeffrey A; Larson, Daniel R; Park, Hye Yoon; Zenklusen, Daniel; Shenoy, Shailesh M; Singer, Robert H

    2014-01-01

    Fluorescent in situ hybridization (FISH) allows the quantification of single mRNAs in budding yeast using fluorescently labeled single-stranded DNA probes, a wide-field epifluorescence microscope and a spot-detection algorithm. Fixed yeast cells are attached to coverslips and hybridized with a mixture of FISH probes, each conjugated to several fluorescent dyes. Images of cells are acquired in 3D and maximally projected for single-molecule analysis. Diffraction-limited labeled mRNAs are observed as bright fluorescent spots and can be quantified using a spot-detection algorithm. FISH preserves the spatial distribution of cellular RNA distribution within the cell and the stochastic fluctuations in individual cells that can lead to phenotypic differences within a clonal population. This information, however, is lost if the RNA content is measured on a population of cells by using reverse transcriptase PCR, microarrays or high-throughput sequencing. The FISH procedure and image acquisition described here can be completed in 3 d. PMID:22301778

  6. Application of locked nucleic acid-based probes in fluorescence in situ hybridization.

    PubMed

    Fontenete, Sílvia; Carvalho, Daniel; Guimarães, Nuno; Madureira, Pedro; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

    2016-07-01

    Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2'-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications of LNA probes for the detection of microorganisms. PMID:26969040

  7. Micro fluorescence in situ hybridization (μFISH) for spatially multiplexed analysis of a cell monolayer.

    PubMed

    Huber, D; Autebert, J; Kaigala, G V

    2016-04-01

    We here present a micrometer-scale implementation of fluorescence in situ hybridization that we term μFISH. This μFISH implementation makes use of a non-contact scanning probe technology, namely, a microfluidic probe (MFP) that hydrodynamically shapes nanoliter volumes of liquid on a surface with micrometer resolution. By confining FISH probes at the tip of this microfabricated scanning probe, we locally exposed approximately 1000 selected MCF-7 cells of a monolayer to perform incubation of probes - the rate-limiting step in conventional FISH. This method is compatible with the standard workflow of conventional FISH, allows re-budgeting of the sample for various tests, and results in a ~ 15-fold reduction in probe consumption. The continuous flow of probes and shaping liquid on these selected cells resulted in a 120-fold reduction of the hybridization time compared with the standard protocol (3 min vs. 6 h) and efficient rinsing, thereby shortening the total FISH assay time for centromeric probes. We further demonstrated spatially multiplexed μFISH, enabling the use of spectrally equivalent probes for detailed and real-time analysis of a cell monolayer, which paves the way towards rapid and automated multiplexed FISH on standard cytological supports. PMID:27138995

  8. Microbial populations identified by fluorescence in situ hybridization in a constructed wetland treating acid coal mine drainage.

    PubMed

    Nicomrat, Duongruitai; Dick, Warren A; Tuovinen, Olli H

    2006-01-01

    Microorganisms are an integral part of the biogeochemical processes in wetlands, yet microbial communities in sediments within constructed wetlands receiving acid mine drainage (AMD) are only poorly understood. The purpose of this study was to characterize the microbial diversity and abundance in a wetland receiving AMD using fluorescence in situ hybridization (FISH) analysis. Seasonal samples of oxic surface sediments, comprised of Fe(III) precipitates, were collected from two treatment cells of the constructed wetland system. The pH of the bulk samples ranged between pH 2.1 and 3.9. Viable counts of acidophilic Fe and S oxidizers and heterotrophs were determined with a most probable number (MPN) method. The MPN counts were only a fraction of the corresponding FISH counts. The sediment samples contained microorganisms in the Bacteria (including the subgroups of acidophilic Fe- and S-oxidizing bacteria and Acidiphilium spp.) and Eukarya domains. Archaea were present in the sediment surface samples at < 0.01% of the total microbial community. The most numerous bacterial species in this wetland system was Acidithiobacillus ferrooxidans, comprising up to 37% of the bacterial population. Acidithiobacillus thiooxidans was also abundant. Heterotrophs in the Acidiphilium genus totaled 20% of the bacterial population. Leptospirillum ferrooxidans was below the level of detection in the bacterial community. The results from the FISH technique from this field study are consistent with results from other experiments involving enumeration by most probable number, dot-blot hybridization, and denaturing gradient gel electrophoresis analyses and with the geochemistry of the site. PMID:16825452

  9. In Situ Carbonized Cellulose-Based Hybrid Film as Flexible Paper Anode for Lithium-Ion Batteries.

    PubMed

    Cao, Shaomei; Feng, Xin; Song, Yuanyuan; Liu, Hongjiang; Miao, Miao; Fang, Jianhui; Shi, Liyi

    2016-01-20

    Flexible free-standing carbonized cellulose-based hybrid film is integrately designed and served both as paper anode and as lightweight current collector for lithium-ion batteries. The well-supported heterogeneous nanoarchitecture is constructed from Li4Ti5O12 (LTO), carbonized cellulose nanofiber (C-CNF) and carbon nanotubes (CNTs) using by a pressured extrusion papermaking method followed by in situ carbonization under argon atmospheres. The in situ carbonization of CNF/CNT hybrid film immobilized with uniform-dispersed LTO results in a dramatic improvement in the electrical conductivity and specific surface area, so that the carbonized paper anode exhibits extraordinary rate and cycling performance compared to the paper anode without carbonization. The flexible, lightweight, single-layer cellulose-based hybrid films after carbonization can be utilized as promising electrode materials for high-performance, low-cost, and environmentally friendly lithium-ion batteries. PMID:26727586

  10. Fluorescent in situ hybridization in routinely processed bone marrow aspirate clot and core biopsy sections.

    PubMed Central

    Miranda, R. N.; Mark, H. F.; Medeiros, L. J.

    1994-01-01

    Fluorescent in situ hybridization (FISH) is a technique which complements conventional cytogenetic banding analysis by allowing the evaluation of cells in interphase as well as metaphase. This technique has been used to study air-dried peripheral blood and bone marrow aspirate smears. We have applied the FISH technique to study routinely processed sections of bone marrow aspirate clot and decalcified core biopsy specimens, fixed in either formalin or B5 and embedded in paraffin. We evaluated 28 specimens (8 aspirate clot and 20 core biopsy sections) for chromosome 8 copy number, studied previously by conventional cytogenetics, and found the following distribution: 15 with disomy, 11 with trisomy, and 2 with tetrasomy. Using a chromosome 8 alpha-satellite probe, we detected fluorescent hybridization signals in 18 of 28 specimens (64%); 6 of 8 (75%) aspirate clot sections, and 12 of 20 (60%) core biopsy sections. Ten of 13 (77%) B5-fixed and 8 of 15 (53%) formalin-fixed specimens had hybridizing signals. Specimen age was a significant factor; 10 of 11 (91%) specimens processed within the last 6 months showed signals, in contrast with 8 of 17 (47%) specimens older than 6 months. In the positive specimens, 200 cells were analyzed in areas where individual cells could be identified. In the disomic specimens, two signals per cell were seen in 34 to 66% of the cells. Rare cells (0-2%) with three signals were detected. In the trisomic specimens, three signals per cell were seen in 19 to 46% of the cells. In the tetrasomic specimens, four signals per cell were seen in 15 to 25% of the cells. We conclude that the FISH technique may be useful in the detection of numerical chromosomal abnormalities such as trisomy and tetrasomy 8 in routinely processed bone marrow aspirate clot and decalcified core biopsy sections. Images Figure 1 Figure 2 Figure 3 PMID:7992836

  11. A methodology for the combined in situ analyses of the precursor and mature forms of microRNAs and correlation with their putative targets

    PubMed Central

    Nuovo, Gerard J; Elton, Terry S; Nana-Sinkam, Patrick; Volinia, Stefano; Croce, Carlo M; Schmittgen, Thomas D

    2009-01-01

    There are relatively few protocols described for the in situ detection of microRNA (miRNA) and they often use cryostat sections, signal amplification and hybridization or washes of 50−60 °C. This protocol describes in situ miRNA detection that can be done in paraffin-embedded, formalin-fixed tissue. Detection of the miRNA precursors can be done by RT in situ PCR, which can theoretically detect one copy per cell. The key variable for the RT in situ PCR protocol is optimal protease digestion, which is then followed by overnight DNase digestion and target specific incorporation of the reported nucleotide into the amplified cDNA. Detection of mature miRNAs is achieved by in situ hybridization with locked nucleic acid probes. This part of the protocol involves a brief protease digestion, followed by an overnight hybridization, short low stringency wash and detection of the labeled probe. The key variables for this method include probe concentration and stringency conditions. Each miRNA in situ method takes 1 d. The final step of the protocol involves colabeling by immunohistochemistry for the putative target of the miRNA, which is done after the in situ hybridization step and takes a few hours. PMID:19131963

  12. In situ evolutionary rate measurements show ecological success of recently emerged bacterial hybrids.

    PubMed

    Denef, Vincent J; Banfield, Jillian F

    2012-04-27

    Few data are available on how quickly free-living microorganisms evolve. We analyzed biofilms collected from a well-defined acid mine drainage system over 9 years to investigate the processes and determine rates of bacterial evolution directly in the environment. Population metagenomic analyses of the dominant primary producer yielded the nucleotide substitution rate, which we used to show that proliferation of a series of recombinant bacterial strains occurred over the past few decades. The ecological success of hybrid bacterial types highlights the role of evolutionary processes in rapid adaptation within natural microbial communities. PMID:22539719

  13. Telomeric IGH Losses Detectable by Fluorescence in Situ Hybridization in Chronic Lymphocytic Leukemia Reflect Somatic VH Recombination Events

    PubMed Central

    Wlodarska, Iwona; Matthews, Christine; Veyt, Ellen; Pospisilova, Helena; Catherwood, Mark A.; Poulsen, Tim S.; Vanhentenrijk, Vera; Ibbotson, Rachel; Vandenberghe, Peter; Morris, T.C.M. “Curly”; Alexander, H. Denis

    2007-01-01

    Routine interphase fluorescence in situ hybridization (FISH) analysis of chronic lymphocytic leukemia (CLL) with LSI IGH/CCND1 assay, applied to differentiate CLL from leukemic mantle cell lymphoma, identified a subset of cases (42/174) with translocation-like IGH signal pattern. To unravel the underlying 14q32/IGH aberrations, 14 of these cases were subjected to cytogenetic, detailed FISH, and VH mutation analyses. FISH identified cryptic losses of various portions of the IGHV region in all 14 cases. Fine mapping of these VH deletions revealed a strict correlation between their distal border and localization of the used VH gene, suggesting that they are not oncogenic but reflect physiological events accompanying somatic V-D-J assembly. This hypothesis was further supported by FISH analysis of 20 CLL and hairy cell leukemia cases with the known VH usage showing a constant loss of sequences proximal to the used gene, identification of VH deletions in normal B cells, and their exclusive demonstration in B cell malignancies, but not of T cell and myeloid linage. Given that these cryptic physiological VH losses in B cells may seriously complicate analysis of B cell leukemia/lymphoma and lead to false conclusions, FISH users should take them into consideration when interpreting IGH aberrations in these malignancies. PMID:17251335

  14. Technical Note: Determination of the metabolically active fraction of benthic foraminifera by means of Fluorescent In Situ Hybridization (FISH)

    NASA Astrophysics Data System (ADS)

    Borrelli, C.; Sabbatini, A.; Luna, G. M.; Nardelli, M. P.; Sbaffi, T.; Morigi, C.; Danovaro, R.; Negri, A.

    2011-08-01

    Benthic foraminifera are an important component of the marine biota, but protocols for investigating their viability and metabolism are still extremely limited. Classical studies on benthic foraminifera have been based on direct counting under light microscopy. Typically, these organisms are stained with Rose Bengal, which binds proteins and other macromolecules, but does not allow discrimination between viable and recently dead organisms. The fluorescent in situ hybridization technique (FISH) represents a new and useful approach to identify living cells possessing an active metabolism. Our work is the first test of the suitability of the FISH technique, based on fluorescent probes targeting the 18S rRNA, to detect live benthic foraminifera. The protocol was applied on Ammonia group and Miliolids, as well as on agglutinated polythalamous (i.e., Leptohalysis scottii and Eggerella scabra) and soft-shelled monothalamous (i.e., Psammophaga sp. and saccamminid morphotypes) taxa. The results from FISH analyses were compared with those obtained, on the same specimens assayed with FISH, from microscopic analysis of the cytoplasm colour, presence of pigments and pseudopodial activity. Our results indicate that FISH targets only metabolically active foraminifera, and allows discerning from low to high cellular activity, validating the hypothesis that the intensity of the fluorescent signal emitted by the probe is dependent upon the physiological status of cells. These findings support the usefulness of this molecular approach as a key tool for obtaining information on the physiology of living foraminifera, both in field and experimental settings.

  15. In-situ sol-gel synthesis and characterization of bioactive pHEMA/SiO2 blend hybrids.

    PubMed

    Silvestri, B; Luciani, G; Costantini, A; Tescione, F; Branda, F; Pezzella, A

    2009-05-01

    A novel procedure to synthesize poly(2-hydroxyethylmethacrylate)-silica blend hybrids is presented. Methacrylate monomers bearing an alkoxysilyl unit, prepared by Michael addition of 2-hydroxyethylmethacrylate (HEMA) to 3-Aminopropyltriethoxysilane (APTS) were employed. By (13)C NMR and mass analysis it was possible to establish the formation of coupling hybrid species. Hybrid materials, with final concentration ranging from 10% to 30% w/w of silica gel to the mass of polymer, were obtained through basic catalyzed sol-gel process of tetraethoxysilane (TEOS) and the alkoxysilyl unit of the hybrid monomer, followed by in-situ free-radical polymerization. The hybrids were characterized as far as concerns their thermal properties (glass transition temperature, decomposition temperature), their sorption behavior in water, and in-vitro bioactivity. Optical transparency, higher glass transition temperature, and higher decomposition temperature than pHEMA suggest an increase in either density or intensity of cross-links between the organic and the inorganic phases. The swelling ratio of the 30% hybrids is comparable to pHEMA, whereas it is lower for the other compositions. In-vitro bioactivity of the hybrids, due to the inorganic phase, was ascertained. Soaking time required for apatite deposition on the samples surface decreases as the content of silica gel increases. Therefore, the obtained bioactive hybrids can be used to make bioactive scaffolds for bone engineering. PMID:18823022

  16. Detection of human papillomavirus type 6/11 DNA in conjunctival papillomas by in situ hybridization with radioactive probes

    SciTech Connect

    McDonnell, P.J.; McDonnell, J.M.; Kessis, T.; Green, W.R.; Shah, K.V.

    1987-11-01

    Twenty-three conjunctival papillomas and 28 conjunctival dysplasias were examined for human papillomavirus (HPV)-DNA sequences by in situ hybridization with nick-translated /sup 35/S-labeled HPV probes. Adjacent paraffin sections were hybridized with HPV type 2, 6, 16, and 18 probes at Tm - 17 degrees C. Fifteen tissues, all papillomas, displayed positive hybridization with the HPV-6 probe. Infection with HPV-6 (or the closely related HPV-11) appeared to be responsible for most of the conjunctival papillomas of children and young adults. The presence of genital tract HPV-6 in these lesions suggests that some of the infections were acquired during passage through an infected birth canal. The lack of hybridization in adult conjunctival dysplasias indicates either that HPVs are not associated with this condition or that the probes and the technique utilized were not adequate for demonstration of this association.

  17. Orbitrap-based mass analyser for in-situ characterization of asteroids: ILMA, Ion Laser Mass Analyser

    NASA Astrophysics Data System (ADS)

    Briois, C.; Cotti, H.; Thirkell, L.; Space Orbitrap Consortium[K. Aradj, French; Bouabdellah, A.; Boukrara, A.; Carrasco, N.; Chalumeau, G.; Chapelon, O.; Colin, F.; Coll, P.; Engrand, C.; Grand, N.; Kukui, A.; Lebreton, J.-P.; Pennanech, C.; Szopa, C.; Thissen, R.; Vuitton, V.; Zapf], P.; Makarov, A.

    2014-07-01

    Since about a decade the boundaries between comets and carbonaceous asteroids are fading [1,2]. No doubt that the Rosetta mission should bring a new wealth of data on the composition of comets. But as promising as it may look, the mass resolving power of the mass spectrometers onboard (so far the best on a space mission) will only be able to partially account for the diversity of chemical structures present. ILMA (Ion-Laser Mass Analyser) is a new generation high mass resolution LDI-MS (Laser Desorption-Ionization Mass Spectrometer) instrument concept using the Orbitrap technique, which has been developed in the frame of the two Marco Polo & Marco Polo-R proposals to the ESA Cosmic Vision program. Flagged by ESA as an instrument concept of interest for the mission in 2012, it has been under study for a few years in the frame of a Research and Technology (R&T) development programme between 5 French laboratories (LPC2E, IPAG, LATMOS, LISA, CSNSM) [3,4], partly funded by the French Space Agency (CNES). The work is undertaken in close collaboration with the Thermo Fisher Scientific Company, which commercialises Orbitrap-based laboratory instruments. The R&T activities are currently concentrating on the core elements of the Orbitrap analyser that are required to reach a sufficient maturity level for allowing design studies of future space instruments. A prototype is under development at LPC2E and a mass resolution (m/Δm FWHM) of 100,000 as been obtained at m/z = 150 for a background pressure of 10^{-8} mbar. ILMA would be a key instrument to measure the molecular, elemental and isotopic composition of objects such as carbonaceous asteroids, comets, or other bodies devoid of atmosphere such as the surface of an icy satellite, the Moon, or Mercury.

  18. Comparison of 35S and biotin as labels for in situ hybridization: Use of an HPV model system

    SciTech Connect

    Unger, E.R.; Hammer, M.L.; Chenggis, M.L. )

    1990-01-01

    Colorimetric in situ hybridization is a method of potential importance in diagnosis and research. The largest criticism of the method has been a perceived loss of sensitivity compared with autoradiographic techniques. Our more positive experience with automation of colorimetric in situ hybridization led us to undertake a direct comparison of the sensitivity of 35S- and biotin-labeled probes. Serial sections of formalin-fixed, paraffin-embedded cell pellets from four human cervical carcinoma cell lines with known copies of HPV (CaSki, 400-600 copies HPV 16; HeLa, 10-50 copies HPV 18; SiHa, 1-2 copies HPV 16; HTB31, no known copies HPV) were hybridized with protocols optimized for autoradiographic or colorimetric detection. Both methods gave comparable results, with differences in each technique seen at the limits of sensitivity. The 1-2 copies of HPV 16 per SiHa cell can be detected with both methods; however, grain counting is required for interpretation of the autoradiographic result. This degree of sensitivity for colorimetric in situ hybridization in formalin-fixed, paraffin-embedded material is achieved through careful optimization of probe size and labeling, adequate tissue digestion, and removal of background. Autoradiography may be preferred in situations where quantitation is required, but colorimetric detection retains the advantages of speed, potential for automation, and improved localization of signal with comparable sensitivity.

  19. Luminescent hybrid lanthanide sulfates and lanthanide sulfonate-carboxylates with 1,10-phenanthroline involving in-situ oxidation of 2-mercaptonbenzoic acid

    SciTech Connect

    Zhong, Jie-Cen; Wan, Fang; Sun, Yan-Qiong; Chen, Yi-Ping

    2015-01-15

    A series of lanthanide sulfates and lanthanide sulfonate-carboxylates, [Ln{sub 2}(phen){sub 2}(SO{sub 4}){sub 3}(H{sub 2}O){sub 2}]{sub n} (I:Ln=Nd(1a), Sm(1b), Eu(1c), phen=1,10-phenanthroline) and [Ln(phen)(2-SBA)(BZA)]{sub n} (II: Ln=Sm(2a), Eu(2b), Dy(2c), 2-SBA=2-sulfobenzoate, BZA=benzoate) have been hydrothermally synthesized from lanthanide oxide, 2-mercaptonbenzoic acid with phen as auxiliary ligand and characterized by single-crystal X-ray diffraction, elemental analyses, IR spectra, TG analyses and luminescence spectroscopy. Interestingly, SO{sub 4}{sup 2−} anions in I came from the in situ deep oxidation of thiol groups of 2-mercaptonbenzoic acid while 2-sulfobenzoate and benzoate ligands in II from the middle oxidation and desulfuration reactions of 2-mercaptonbenzoic acid. Compounds I are organic–inorganic hybrid lanthanide sulfates, which have rare one-dimensional column-like structures. Complexes II are binuclear lanthanide sulfonate-carboxylates with 2-sulfobenzoate and benzoate as bridges and 1,10-phenanthroline as terminal. Photoluminescence studies reveal that complexes I and II exhibit strong lanthanide characteristic emission bands in the solid state at room temperature. - Graphical abstract: Lanthanide sulfates and lanthanide sulfonate-carboxylates have been hydrothermally synthesized. Interestingly, sulfate anions, 2-sulfobenzoate and benzoate ligands came from the in situ oxidation and desulfuration reactions of 2-mercaptonbenzoic acid. - Highlights: • In situ oxidation and desulfuration reactions of 2-mercaptonbenzoic acid. • The organic–inorganic hybrid lanthanide sulfates with one-dimensional column-like structure. • The dinuclear lanthanide sulfonate-carboxylates. • The emission spectra exhibit the characteristic transition of {sup 5}D{sub 0}→{sup 7}F{sub J} (J=0–4) of the Eu(III)

  20. [Chromosomal structure of the hybrids between Allium cepa L. and Allium fistulosum L. with relative resistance to downy mildew based on in situ hybridization].

    PubMed

    Budylin, M V; Kan, L Iu; Romanov, V S; Khrustaleva, L I

    2014-04-01

    Genomic in situ hybridization (GISH) was used for a chromosomal composition study of the later generations of interspecific hybrids between A. cepa L. and A. fistulosum L., which are relatively resistant to downy mildew (peronosporosis). GISH revealed that F2 hybrids, which did not produce seeds, were triploids (2n = 3x = 24) with 24 chromosomes and possessed in their compliments 16 chromosomes of A. fistulosum L. and eight chromosomes of A. cepa L. or eight chromosomes of A. fistulosum L. and 16 chromosomes of A. cepa L. The advanced F5 hybrid, which produced few seeds, was amphidiploid with 32 chromosomes. BC1F5 hybrid was triploid with eight chromosomes of A. fistulosum L. and 16 chromosomes of A. cepa L., which did not produce seeds. BC2 (BC1F5) plant was amphidiploid that possessed 4 recombinant chromosomes and produced few seeds. GISH results point to 2n-gametes formation in macro- and microsporogenesis of the hybrids. The mechanism of 2n-gametes formation and the possibility of apomixes events in the backcrossing progeny are discussed. PMID:25715446

  1. In situ hybridization to cytogenetic bands of yeast artificial chromosomes covering 50% of human Xq24-Xq28 DNA

    PubMed Central

    Montanaro, Vittorio; Casamassimi, Amelia; D'Urso, Michele; Yoon, Jae-Young; Freije, Wadiha; Schlessinger, David; Muenke, Maximilian; Nussbaum, Robert L.; Saccone, Salvatore; Maugeri, Silvana; Santoro, Anna Maria; Motta, Salvatore; Della Valle, Giuliano

    1991-01-01

    From the collection described by Abidi et al., 102 yeast artificial chromosomes (YACs) with human DNA inserts more than 300 kb in length were assigned to chromosomal band positions on early metaphase chromosomes by in situ hybridization using the biotin-avidin method. All the YACs hybridized within the Xq24-Xqter region, supporting the origin of the vast majority of the YACs from single human X-chromosomal sites. With assignments precise to ±0.5 bands, YACs were distributed among cytogenetic bands to roughly equal extents. Thus, there is no gross bias in the cloning of DNA from different bands into large YACs. To test band assignments further, hybridizations were carried out blind, and band positions were then compared with (1) probe localizations in cases in which a reported location was present in one of the YACs; (2) cross-hybridization of a labeled YAC with others in the collection; and (3) hybridization to a panel of DNAs from a series of hybrid cells containing Xq DNA truncated at various regions. Of 31 cases in which YACs contained a probe with a previously reported location, 28 in situ assignments were in agreement, and 14 other assignments, including one of the three discordant with probe localization, were confirmed by YAC cross-hybridization studies. Results with a group of nine YACs were further confirmed with a panel of somatic cell hybrid DNAs from that region. Five YACs hybridized both to Xq25 and to a second site (four in Xq27 and one in Xq28), suggestive of some duplication of DNA of the hybrid cell and perhaps in normal X chromosomes. The in situ assignments are thus sufficient to place YACs easily and systematically within bins of about 7–10 Mb and to detect some possible anomalies. Furthermore, on the basis of expectations for random cloning of DNA in YACs, the assigned YACs probably cover more than 50% of the total Xq24-Xq28 region. This provides one way to initiate the assembly of YAC contigs over extended chromosomal regions. Images

  2. Porous titania/carbon hybrid microspheres templated by in situ formed polystyrene colloids.

    PubMed

    Cheng, Ting; Zhang, Guoqiang; Xia, Yonggao; Sun, Zaicheng; Yang, Zhaohui; Liu, Rui; Xiao, Ying; Wang, Xiaoyan; Wang, Meimei; Ban, Jianzhen; Yang, Liangtao; Ji, Qing; Qiu, Bao; Chen, Guoxin; Chen, Huifeng; Lin, Yichao; Pei, Xiaoying; Wu, Qiang; Meng, Jian-Qiang; Liu, Zhaoping; Chen, Liang; Xiao, Tonghu; Sun, Ling-Dong; Yan, Chun-Hua; Butt, Hans Jürgen; Cheng, Ya-Jun

    2016-05-01

    A new strategy to synthesize hierarchical, porous titania/carbon (TiO2/C) hybrid microspheres via solvothermal reaction in N,N'-dimethyl formamide (DMF) has been developed. In situ formed polystyrene (PS) colloids have been used as templating agent and carbon source, through which TiO2/PS microspheres with a diameter of ca. 1 μm are built by packed TiO2 nanoparticles of tens of nanometers. The TiO2/PS microspheres are converted to TiO2/C microspheres with different amounts of carbon under controlled calcination condition. The mechanism investigation unveils that the introduction of concentrated HCl creates surface tension between PS and DMF, leading to the formation of PS colloids in solution. The solvothermal treatment further promotes the formation of PS colloids and integration of the titania nanoparticles within the PS colloids. The morphology, crystallinity, nature and content of carbon, UV-Vis absorption, carbon doping, pore size distribution, pore volume, and BET surface area of the TiO2 microspheres with different amounts of carbon have been measured. The applications of the TiO2/C hybrid microspheres as photo catalyst for water splitting and lithium-ion battery anode have been demonstrated. Superior photo catalytic activity for hydrogen conversion under both full spectrum and visible light illumination compared to commercial P25 has been observed for the TiO2/C microspheres with 2 wt% of carbon. Besides, the TiO2/C microspheres with 8 wt% of carbon as lithium-ion battery anode showed a much higher capacity than the bare TiO2 microsphere anode. The origin for the enhanced performance as photo catalyst and lithium-ion battery anode is discussed. PMID:26896772

  3. A novel triple-color detection procedure for brightfield microscopy, combining in situ hybridization with immunocytochemistry.

    PubMed

    Speel, E J; Jansen, M P; Ramaekers, F C; Hopman, A H

    1994-10-01

    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. PMID:7930513

  4. Comparative cytogenetic characterization of primary canine melanocytic lesions using array CGH and fluorescence in situ hybridization.

    PubMed

    Poorman, Kelsey; Borst, Luke; Moroff, Scott; Roy, Siddharth; Labelle, Philippe; Motsinger-Reif, Alison; Breen, Matthew

    2015-06-01

    Melanocytic lesions originating from the oral mucosa or cutaneous epithelium are common in the general dog population, with up to 100,000 diagnoses each year in the USA. Oral melanoma is the most frequent canine neoplasm of the oral cavity, exhibiting a highly aggressive course. Cutaneous melanocytomas occur frequently, but rarely develop into a malignant form. Despite the differential prognosis, it has been assumed that subtypes of melanocytic lesions represent the same disease. To address the relative paucity of information about their genomic status, molecular cytogenetic analysis was performed on the three recognized subtypes of canine melanocytic lesions. Using array comparative genomic hybridization (aCGH) analysis, highly aberrant distinct copy number status across the tumor genome for both of the malignant melanoma subtypes was revealed. The most frequent aberrations included gain of dog chromosome (CFA) 13 and 17 and loss of CFA 22. Melanocytomas possessed fewer genome wide aberrations, yet showed a recurrent gain of CFA 20q15.3-17. A distinctive copy number profile, evident only in oral melanomas, displayed a sigmoidal pattern of copy number loss followed immediately by a gain, around CFA 30q14. Moreover, when assessed by fluorescence in situ hybridization (FISH), copy number aberrations of targeted genes, such as gain of c-MYC (80 % of cases) and loss of CDKN2A (68 % of cases), were observed. This study suggests that in concordance with what is known for human melanomas, canine melanomas of the oral mucosa and cutaneous epithelium are discrete and initiated by different molecular pathways. PMID:25511566

  5. Microfluidic fluorescence in situ hybridization and flow cytometry (µFlowFISH)

    PubMed Central

    Liu, Peng; Meagher, Robert J.; Light, Yooli Kim; Yilmaz, Suzan; Chakraborty, Romy; Arkin, Adam P.; Hazen, Terry C.; Singh, Anup K.

    2011-01-01

    We describe an integrated microfluidic device (µFlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(VI) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The µFlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed; and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp. strain RCH2 that are involved in Cr(VI) reduction and immobilization. Combined labeling and detection efficiencies of 74–97% were observed in experiments with simple mixtures of cultured cells confirmed specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of µFlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford Site. We were able to monitor the numbers of Pseudomonas sp. with only 100–200 cells loaded into the microchip. The µFlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome. PMID:21755095

  6. Multiplex fluorescence in situ hybridization (M-FISH) and confocal laser scanning microscopy (CLSM) to analyze multispecies oral biofilms.

    PubMed

    Karygianni, Lamprini; Hellwig, Elmar; Al-Ahmad, Ali

    2014-01-01

    Multiplex fluorescence in situ hybridization (M-FISH) constitutes a favorable microbiological method for the analysis of spatial distribution of highly variable phenotypes found in multispecies oral biofilms. The combined use of confocal laser scanning microscopy (CLSM) produces high-resolution three-dimensional (3D) images of individual bacteria in their natural environment. Here, we describe the application of M-FISH on early (Streptococcus spp., Actinomyces naeslundii) and late colonizers (Fusobacterium nucleatum, Veillonella spp.) of in situ-formed oral biofilms, the acquisition of CLSM images, as well as the qualitative and quantitative analysis of these digitally obtained and processed images. PMID:24664826

  7. Development of a PNA Probe for Fluorescence In Situ Hybridization Detection of Prorocentrum donghaiense

    PubMed Central

    Chen, Guofu; Zhang, Chunyu; Zhang, Baoyu; Wang, Guangce; Lu, Douding; Xu, Zhong; Yan, Peishen

    2011-01-01

    Prorocentrum donghaiense is a common but dominant harmful algal bloom (HAB) species, which is widely distributed along the China Sea coast. Development of methods for rapid and precise identification and quantification is prerequisite for early-stage warning and monitoring of blooms due to P. donghaiense. In this study, sequences representing the partial large subunit rDNA (D1–D2), small subunit rDNA and internal transcribed spacer region (ITS-1, 5.8S rDNA and ITS-2) of P. donghaiense were firstly obtained, and then seven candidate DNA probes were designed for performing fluorescence in situ hybridization (FISH) tests on P. donghaiense. Based on the fluorescent intensity of P. donghaiense cells labeled by the DNA probes, the probe DP0443A displayed the best hybridization performance. Therefore, a PNA probe (PP0443A) analogous to DP0443A was used in the further study. The cells labeled with the PNA probe displayed more intensive green fluorescence than that labeled with its DNA analog. The PNA probe was used to hybridize with thirteen microalgae belonging to five families, i.e., Dinophyceae, Prymnesiophyceae, Raphidophyceae, Chlorophyceae and Bacillariophyceae, and showed no visible cross-reaction. Finally, FISH with the probes PP0443A and DP0443A and light microscopy (LM) analysis aiming at enumerating P. donghaiense cells were performed on the field samples. Statistical comparisons of the cell densities (cells/L) of P. donghaiense in the natural samples determined by FISH and LM were performed using one-way ANOVA and Duncan's multiple comparisons of the means. The P. donghaiense cell densities determined by LM and the PNA probe are remarkably higher than (p<0.05) that determined by the DNA probe, while no significant difference is observed between LM and the PNA probe. All results suggest that the PNA probe is more sensitive that its DNA analog, and therefore is promising for the monitoring of harmful algal blooms of P. donghaiense in the future. PMID:22022408

  8. Assessment of retrospective dose estimation, with fluorescence in situ hybridization (FISH), of six victims previously exposed to accidental ionizing radiation.

    PubMed

    Liu, Qing-Jie; Lu, Xue; Zhao, Xiao-Tao; Feng, Jiang-Bin; Lü, Yu-Min; Jiang, En-Hai; Zhang, Shu-Lan; Chen, De-Qing; Jia, Ting-Zhen; Liang, Li

    2014-01-01

    The present study aims to evaluate the use of the fluorescence in situ hybridization (FISH) translocation assay for retrospective dose estimation of acute accidental exposure to radiation in the past. Reciprocal translocation analysis by FISH with three whole-chromosome probes was performed on normal peripheral blood samples. Samples were irradiated with 0-5Gy (60)Co γ-rays in vitro, and dose-effect curves were established. FISH-based translocation analyses for six accident victims were then performed, and biological doses were estimated retrospectively by comparison with the dose-effect curves. Reconstructed doses by FISH were compared with estimated doses obtained by analysis of di-centrics performed soon after exposure, or with dose estimates from tooth-enamel electron paramagnetic resonance (EPR) data obtained at the same time as the FISH analysis. Follow-up FISH analyses for an adolescent victim were performed. Results showed that dose-effect curves established in the present study follow a linear-quadratic model, regardless of the background translocation frequency. Estimated doses according to two dose-effect curves for all six victims were similar. FISH dose estimations of three adult victims exposed to accidental radiation less than a decade prior to analysis (3, 6, or 7 years ago) were consistent with those estimated with tooth-enamel EPR measurements or analyses of di-centrics. Estimated doses of two other adult victims exposed to radiation over a decade prior to analysis (16 or 33 years ago) were underestimated and two to three times lower than the values obtained from analysis of di-centrics or tooth-enamel EPR. Follow-up analyses of the adolescent victim showed that doses estimated by FISH analysis decrease rapidly over time. Therefore, the accuracy of dose estimates by FISH is acceptable only when analysis is performed less than 7 years after exposure. Measurements carried out more than a decade after exposure through FISH analysis resulted in

  9. Imaging of multiple mRNA targets using quantum dot based in situ hybridization and spectral deconvolution in clinical biopsies

    SciTech Connect

    Tholouli, Eleni; Hoyland, Judith A.; Di Vizio, Dolores; O'Connell, Fionnuala; MacDermott, Sarah A.; Twomey, David; Levenson, Richard; Yin, John A. Liu; Golub, Todd R.; Loda, Massimo; Byers, Richard . E-mail: r.byers@manchester.ac.uk

    2006-09-22

    Gene expression mapping using microarray analysis has identified useful gene signatures for predicting outcome. However, little of this has been translated into clinically effective diagnostic tools as microarrays require high quality fresh-frozen tissue samples. We describe a methodology of multiplexed in situ hybridization (ISH) using a novel combination of quantum dot (QD)-labeled oligonucleotide probes and spectral imaging analysis in routinely processed, formalin-fixed paraffin embedded human biopsies. The conditions for QD-ISH were optimized using a poly d(T) oligonucleotide in decalcified bone marrow samples. Single and multiplex QD-ISH was performed in samples with acute leukemia and follicular lymphoma using oligonucleotide probes for myeloperoxidase, bcl-2, survivin, and XIAP. Spectral imaging was used for post hybridization tissue analysis, enabling separation of spatially colocalized signals. The method allows quantitative characterization of multiple gene expression using non-bleaching fluorochromes. This is expected to facilitate multiplex in situ transcript detection in routinely processed human clinical tissue.

  10. Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization

    PubMed Central

    Williamson, Iain; Berlivet, Soizik; Eskeland, Ragnhild; Boyle, Shelagh; Illingworth, Robert S.; Paquette, Denis

    2014-01-01

    Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods. PMID:25512564

  11. Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Malaria in Endemic Areas

    PubMed Central

    Shah, Jyotsna; Mark, Olivia; Weltman, Helena; Barcelo, Nicolas; Lo, Wai; Wronska, Danuta; Kakkilaya, Srinivas; Rao, Aravinda; Bhat, Shalia T.; Sinha, Ruchi; Omar, Sabah; Moro, Manuel; Gilman, Robert H.; Harris, Nick

    2015-01-01

    Malaria is a responsible for approximately 600 thousand deaths worldwide every year. Appropriate and timely treatment of malaria can prevent deaths but is dependent on accurate and rapid diagnosis of the infection. Currently, microscopic examination of the Giemsa stained blood smears is the method of choice for diagnosing malaria. Although it has limited sensitivity and specificity in field conditions, it still remains the gold standard for the diagnosis of malaria. Here, we report the development of a fluorescence in situ hybridization (FISH) based method for detecting malaria infection in blood smears and describe the use of an LED light source that makes the method suitable for use in resource-limited malaria endemic countries. The Plasmodium Genus (P-Genus) FISH assay has a Plasmodium genus specific probe that detects all five species of Plasmodium known to cause the disease in humans. The P. falciparum (PF) FISH assay and P. vivax (PV) FISH assay detect and differentiate between P. falciparum and P. vivax respectively from other Plasmodium species. The FISH assays are more sensitive than Giemsa. The sensitivities of P-Genus, PF and PV FISH assays were found to be 98.2%, 94.5% and 98.3%, respectively compared to 89.9%, 83.3% and 87.9% for the detection of Plasmodium, P. falciparum and P. vivax by Giemsa staining respectively. PMID:26333092

  12. Detection of circovirus infection in pigeons by in situ hybridization using cloned DNA probes.

    PubMed

    Smyth, J A; Weston, J; Moffett, D A; Todd, D

    2001-11-01

    Degenerate primers were designed based on known sequence information for the circoviruses psittacine beak and feather disease virus and porcine circovirus and applied by polymerase chain reaction (PCR) to known virus-infected bursa of Fabricius (BF) from a pigeon. A 548-bp DNA fragment was amplified and shown to be specific to a novel circovirus, named pigeon circovirus (PiCV), and was used to produce sensitive and specific probes for detection of circovirus DNA by in situ hybridization (ISH). Using ISH on BF from 107 pigeons submitted for necropsy, infection was detected in 89%, compared with a histologic detection rate of 66%. Using the ISH technique, infected cells were also found in liver, kidney, trachea, lung, brain, crop, intestine, spleen, bone marrow, and heart of some birds. Large quantities of DNA were present in some of these tissues, and in the absence of BF, liver in particular is identified as a potentially useful organ to examine for presence of PiCV. This high prevalence of infection in diseased birds is noteworthy, emphasizing the need for studies to determine the precise role of this virus as a disease-producing agent. PMID:11724137

  13. Specific Detection of Two Divergent Simian Arteriviruses Using RNAscope In Situ Hybridization

    PubMed Central

    Yú, Shuǐqìng; Caì, Yíngyún; Lyons, Cassandra; Johnson, Reed F.; Postnikova, Elena; Mazur, Steven; Johnson, Joshua C.; Radoshitzky, Sheli R.; Bailey, Adam L.; Lauck, Michael; Goldberg, Tony L.; O’Connor, David H.; Jahrling, Peter B.; Friedrich, Thomas C.; Kuhn, Jens H.

    2016-01-01

    Simian hemorrhagic fever (SHF) is an often lethal disease of Asian macaques. Simian hemorrhagic fever virus (SHFV) is one of at least three distinct simian arteriviruses that can cause SHF, but pathogenesis studies using modern methods have been scarce. Even seemingly straightforward studies, such as examining viral tissue and cell tropism in vivo, have been difficult to conduct due to the absence of standardized SHFV-specific reagents. Here we report the establishment of an in situ hybridization assay for the detection of SHFV and distantly related Kibale red colobus virus 1 (KRCV-1) RNA in cell culture. In addition, we detected SHFV RNA in formalin-fixed, paraffin-embedded tissues from an infected rhesus monkey (Macaca mulatta). The assay is easily performed and can clearly distinguish between SHFV and KRCV-1. Thus, if further developed, this assay may be useful during future studies evaluating the mechanisms by which a simian arterivirus with a restricted cell tropism can cause a lethal nonhuman primate disease similar in clinical presentation to human viral hemorrhagic fevers. PMID:26963736

  14. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method

    PubMed Central

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6–99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  15. Specific Detection of Two Divergent Simian Arteriviruses Using RNAscope In Situ Hybridization.

    PubMed

    Yú, Shu Qìng; Caì, Yíngyún; Lyons, Cassandra; Johnson, Reed F; Postnikova, Elena; Mazur, Steven; Johnson, Joshua C; Radoshitzky, Sheli R; Bailey, Adam L; Lauck, Michael; Goldberg, Tony L; O'Connor, David H; Jahrling, Peter B; Friedrich, Thomas C; Kuhn, Jens H

    2016-01-01

    Simian hemorrhagic fever (SHF) is an often lethal disease of Asian macaques. Simian hemorrhagic fever virus (SHFV) is one of at least three distinct simian arteriviruses that can cause SHF, but pathogenesis studies using modern methods have been scarce. Even seemingly straightforward studies, such as examining viral tissue and cell tropism in vivo, have been difficult to conduct due to the absence of standardized SHFV-specific reagents. Here we report the establishment of an in situ hybridization assay for the detection of SHFV and distantly related Kibale red colobus virus 1 (KRCV-1) RNA in cell culture. In addition, we detected SHFV RNA in formalin-fixed, paraffin-embedded tissues from an infected rhesus monkey (Macaca mulatta). The assay is easily performed and can clearly distinguish between SHFV and KRCV-1. Thus, if further developed, this assay may be useful during future studies evaluating the mechanisms by which a simian arterivirus with a restricted cell tropism can cause a lethal nonhuman primate disease similar in clinical presentation to human viral hemorrhagic fevers. PMID:26963736

  16. Morphing hybrid honeycomb (MOHYCOMB) with in situ Poisson’s ratio modulation

    NASA Astrophysics Data System (ADS)

    Heath, Callum J. C.; Neville, Robin M.; Scarpa, Fabrizio; Bond, Ian P.; Potter, Kevin D.

    2016-08-01

    Electrostatic adhesion can be used as a means of reversible attachment. Through application of high voltage (~2 kV) across closely spaced parallel plate electrodes, significant shear stresses (11 kPa) can be generated. The highest levels of electrostatic holding force can be achieved through close contact of connection surfaces; this is facilitated by flexible electrodes which can conform to reduce air gaps. Cellular structures are comprised of thin walled elements, making them ideal host structures for electrostatic adhesive elements. The reversible adhesion provides control of the internal connectivity of the cellular structure, and determines the effective cell geometry. This would offer variable stiffness and control of the effective Poisson’s ratio of the global cellular array. Using copper-polyimide thin film laminates and PVDF thin film dielectrics, double lap shear electrostatic adhesive elements have been introduced to a cellular geometry. By activating different groups of reversible adhesive interfaces, the cellular array can assume four different cell configurations. A maximum stiffness modulation of 450% between the ‘All off’ and ‘All on’ cell morphologies has been demonstrated. This structure is also capable of in situ effective Poisson’s ratio variations, with the ability to switch between values of ‑0.45 and 0.54. Such a structure offers the potential for tuneable vibration absorption (due to its variable stiffness properties), or as a smart honeycomb with controllable curvature and is termed morphing hybrid honeycomb.

  17. MMP activity in the hybrid layer detected with in situ zymography.

    PubMed

    Mazzoni, A; Nascimento, F D; Carrilho, M; Tersariol, I; Papa, V; Tjäderhane, L; Di Lenarda, R; Tay, F R; Pashley, D H; Breschi, L

    2012-05-01

    Dentinal proteases are believed to play an important role in the degradation of hybrid layers (HL). This study investigated the HL gelatinolytic activity by in situ zymography and functional enzyme activity assay. The hypotheses were that HLs created by an etch-and-rinse adhesive exhibit active gelatinolytic activity, and MMP-2 and -9 activities in dentin increase during adhesive procedures. Etched-dentin specimens were bonded with Adper Scotchbond 1XT and restored with composite. Adhesive/dentin interface slices were placed on microscope slides, covered with fluorescein-conjugated gelatin, and observed with a multi-photon confocal microscope after 24 hrs. Human dentin powder aliquots were prepared and assigned to the following treatments: A, untreated; B, etched with 10% phosphoric acid; or C, etched with 10% phosphoric acid and mixed with Scotchbond 1XT. The MMP-2 and -9 activities of extracts of dentin powder were measured with functional enzyme assays. Intense and continuous enzyme activity was detected at the bottom of the HL, while that activity was more irregular in the upper HL. Both acid-etching and subsequent adhesive application significantly increased MMP-2 and -9 activities (p < 0.05). The results demonstrate, for the first time, intrinsic MMP activity in the HL, and intense activation of matrix-bound MMP activity with both etching and adhesive application. PMID:22354448

  18. c-fos oncogene underexpression in salivary gland tumors as measured by in situ hybridization.

    PubMed Central

    Birek, C.; Lui, E.; Dardick, I.

    1993-01-01

    Tissue from 35 salivary gland tumors and 14 normal salivary glands was analyzed by in situ hybridization and computer-assisted morphometry for the expression of the c-fos oncogene. The normal salivary gland tissues were found to express c-fos focally, mainly in the acinar secretory cells. The majority of the cells in the normal tissues showed a high level of expression (47.74 +/- 5.31% of cells had 46 to 60 grains per cell and another 45.79 +/- 2.18% showed > 60 grains per cell). All the tumors examined exhibited a relatively low, uniform distribution of c-fos expression. For example, in the poorly differentiated adenocarcinomas, 96.83 +/- 04% of the cells were found to have < 15 grains per cell. A general linear model for multivariate analysis showed a significant difference between the various tumor types and the normal salivary gland tissues (P = 0.0001). These data support the hypothesis that salivary gland tumors belong to a group of epithelial neoplasias in which the loss of cellular differentiation is linked with underexpression of the c-fos oncogene. Images Figure 1 Figure 2 Figure 3 PMID:8456948

  19. Just cool it! Cryoprotectant anti-freeze in immunocytochemistry and in situ hybridization.

    PubMed

    Hoffman, Gloria E; Le, Wei Wei

    2004-03-01

    Immunohistochemical techniques offer specificity as well as flexibility for visualizing antigens. Their use with freely floating sections provides a high signal-to-noise ratio and has become a gold standard for brain and a number of other tissues. Yet this approach initially suffered from inability to keep the antigenicity in tissue sections and required immediate processing of all cut sections. Use of sucrose solutions enabled storage at refrigerator temperatures for a few days but longer-term storage was risky and either bacterial/fungal growth or evaporation of the storage solution compromised the integrity of the tissue. Our discovery 25 years ago that tissue sections can be stored for many years at -20 degrees C in an anti-freeze cryoprotectant solution with no loss of antigenicity solved this problem and has become widely used. More recently the utility of tissue stored for many years in anti-freeze cryoprotectant was pushed to new levels by testing new non-radioactive in situ hybridization (ISH) techniques that are based on modern immunocytochemistry. This review touches upon these advances in immunocytochemical technology using examples from neuroscience applications. PMID:15134865

  20. Identification of Marteilia refringens infecting the razor clam Solen marginatus by PCR and in situ hybridization.

    PubMed

    López-Flores, Inmaculada; Garrido-Ramos, Manuel A; de la Herran, Roberto; Ruiz-Rejón, Carmelo; Ruiz-Rejón, Manuel; Navas, José I

    2008-06-01

    Marteilia refringens is a protozoan parasite recognized as a significant pathogen of the European flat oyster Ostrea edulis. It is believed to have a complex life-cycle involving several hosts. In this study, we applied molecular approaches to identify this parasite in samples of the razor clam Solen marginatus from the south west coast of Spain. We used a PCR assay to amplify a fragment of the IGS rDNA region. PCR products were sequenced and the phylogenetic affinity of the sequences was determined. In situ hybridization analysis showed tissue distribution and presence of different developmental stages of the parasite in the digestive diverticula epithelium, which suggested a true parasitism in these individuals. This is the first report of the occurrence of M. refringens in the razor clam S. marginatus in the south Atlantic. The methodology described herein may be useful for accurate identification of the parasite strain in different hosts and thus provide valuable information for marteiliosis control programmes. PMID:18378424

  1. Fifty probands with extra structurally abnormal chromosomes characterized by fluorescence in situ hybridization

    SciTech Connect

    Blennow, E.; Telenius, H.; Nordenskjoeld, M.

    1995-01-02

    Extra structurally abnormal chromosomes (ESACs) are small supernumerary chromosomes often associated with developmental abnormalities and malformations. We present 50 probands with ESACs characterized by fluorescence in situ hybridization using centromere-specific probes and chromosome-specific libraries. ESAC-specific libraries were constructed by flow sorting and subsequent amplification by DOP-PCR. Using such ESAC-specific libraries we were able to outline the chromosome regions involved. Twenty-three of the 50 ESACs were inverted duplications of chromosome 15 (inv dup(15)), including patients with normal phenotypes and others with similar clinical symptoms. These 2 groups differed in size and shape of the inv dup(15). Patients with a large inv dup(15), which included the Prader-Willi region, had a high risk of abnormality, whereas patients with a small inv dup(15), not including the Prader-Willi region, were normal. ESACs derived from chromosomes 13 or 21 appeared to have a low risk of abnormality, while one out of 3 patients with an ESAC derived from chromosome 14 had discrete symptoms. One out of 3 patients with an ESAC derived from chromosome 22 had severe anomalies, corresponding to some of the manifestations of the cat eye syndrome. Small extra ring chromosomes of autosomal origin and ESACs identified as i(12p) or i(18p) were all associated with a high risk of abnormality. 42 refs., 2 figs., 2 tabs.

  2. Visualizing the Spatial Relationship of the Genome with the Nuclear Envelope Using Fluorescence In Situ Hybridization.

    PubMed

    Clements, Craig S; Bikkul, Ural; Ahmed, Mai Hassan; Foster, Helen A; Godwin, Lauren S; Bridger, Joanna M

    2016-01-01

    The genome has a special relationship with the nuclear envelope in cells. Much of the genome is anchored at the nuclear periphery, tethered by chromatin binding proteins such nuclear lamins and other integral membrane proteins. Even though there are global assays such as DAM-ID or ChIP to assess what parts of the genome are associated with the nuclear envelope, it is also essential to be able to visualize regions of the genome in order to reveal their individual relationships with nuclear structures in single cells. This is executed by fluorescence in situ hybridization (FISH) in 2-dimensional flattened nuclei (2D-FISH) or 3-dimensionally preserved cells (3D-FISH) in combination with indirect immunofluorescence to reveal structural proteins. This chapter explains the protocols for 2D- and 3D-FISH in combination with indirect immunofluorescence and discusses options for image capture and analysis. Due to the nuclear envelope proteins being part of the non-extractable nucleoskeleton, we also describe how to prepare DNA halos through salt extraction and how they can be used to study genome behavior and association when combined with 2D-FISH. PMID:27147055

  3. Potential clinical impact of three-dimensional visualization for fluorescent in situ hybridization image analysis

    NASA Astrophysics Data System (ADS)

    Li, Zheng; Li, Shibo; Bin, Zheng; Zhang, Roy; Li, Yuhua; Tian, Huimin; Chen, Wei; Liu, Hong

    2012-05-01

    Chromosomal translocation is strong indication of cancers. Fluorescent in situ hybridization (FISH) can effectively detect this translocation and achieve high accuracy in disease diagnosis and prognosis assessment. For this purpose, whole chromosome paint probes are utilized to image the configuration of DNA fragments. Although two-dimensional (2-D) microscopic images are typically used in FISH signal analysis, we present a case where the translocation occurs in the depth direction where two probed FISH signals are overlapped in the projected image plane. Thus, the translocation cannot be identified. However, when imaging the whole specimen with a confocal microscope at 27 focal planes with 0.5-μm step interval, the translocation can be clearly identified due to the free rotation capability by the three-dimensional (3-D) visualization. Such a translocation detection error of using 2-D images might be critical in detecting and diagnosing early or subtle disease cases where detecting a small number of abnormal cells can make diagnostic difference. Hence, the underlying implication of this report suggests that utilizing 3-D visualization may improve the overall accuracy of FISH analysis for some clinical cases. However, the clinical efficiency and cost of using 3-D versus 2-D imaging methods are also to be assessed carefully.

  4. The design of a microscopic system for typical fluorescent in-situ hybridization applications

    NASA Astrophysics Data System (ADS)

    Yi, Dingrong; Xie, Shaochuan

    2013-12-01

    Fluorescence in situ hybridization (FISH) is a modern molecular biology technique used for the detection of genetic abnormalities in terms of the number and structure of chromosomes and genes. The FISH technique is typically employed for prenatal diagnosis of congenital dementia in the Obstetrics and Genecology department. It is also routinely used to pick up qualifying breast cancer patients that are known to be highly curable by the prescription of Her2 targeted therapy. During the microscopic observation phase, the technician needs to count typically green probe dots and red probe dots contained in a single nucleus and calculate their ratio. This procedure need to be done to over hundreds of nuclei. Successful implementation of FISH tests critically depends on a suitable fluorescent microscope which is primarily imported from overseas due to the complexity of such a system beyond the maturity of the domestic optoelectrical industry. In this paper, the typical requirements of a fluorescent microscope that is suitable for FISH applications are first reviewed. The focus of this paper is on the system design and computational methods of an automatic florescent microscopy with high magnification APO objectives, a fast spinning automatic filter wheel, an automatic shutter, a cooled CCD camera used as a photo-detector, and a software platform for image acquisition, registration, pseudo-color generation, multi-channel fusing and multi-focus fusion. Preliminary results from FISH experiments indicate that this system satisfies routine FISH microscopic observation tasks.

  5. Hybrid Al + Al3Ni metallic foams synthesized in situ via laser engineered net shaping

    NASA Astrophysics Data System (ADS)

    Zheng, Baolong; Li, Ying; Smugeresky, John E.; Zhou, Yizhang; Baker, Dean; Lavernia, Enrique J.

    2011-09-01

    A hybrid, Al + Al3Ni metallic foam was synthesized in situ via laser engineered net shaping (LENS®) of Ni-coated 6061 Al powder in the absence of a foaming agent. During LENS® processing, the Ni coating reacted with the Al matrix, resulting in the simultaneous formation of a fine dispersion of Al3Ni, and a high volume fraction of porosity. As a reinforcement phase, the intermetallic compound formed particles with a size range of 1-5 µm and a volume fraction of 63%, with accompanying 35-300 µm pores with a 60% volume fraction. The microstructure of the as-deposited Al + Al3Ni composite foams was characterized using SEM, EDS, XRD and TEM/HRTEM techniques. The evolution of the microstructure was analyzed on the basis of the thermal field present during deposition, paying particular attention to the thermodynamics of the Al3Ni intermetallic compound formation as well as discussing the mechanisms that may be responsible for the observed porosity. The mechanical behavior of the as-deposited material was characterized using compression and microhardness testing, indicating that the yield strength and hardness are 190 MPa and 320 HV, respectively, which represents an increase of over three times higher than that of annealed Al6061, or similar to heat-treated Al6061 fully dense matrix, and much higher than those of traditional Al alloy foams, and with a low density of 1.64 g/m3.

  6. Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria.

    PubMed Central

    Lange, J L; Thorne, P S; Lynch, N

    1997-01-01

    Current limitations in the methodology for enumeration and identification of airborne bacteria compromise the precision and accuracy of bioaerosol exposure assessment. In this study, flow cytometry and fluorescent in situ hybridization (FISH) were evaluated for the assessment of exposures to airborne bacteria. Laboratory-generated two-component bioaerosols in exposures chambers and complex native bioaerosols in swine barns were sampled with two types of liquid impingers (all-glass impinger-30 and May 3-stage impinger). Aliquots of collection media were processed and enumerated by a standard culture technique, microscopy, or flow cytometry after nucleic acid staining with 4',6-diamidino-2-phenylindole (DAPI) and identified taxonomically by FISH. DAPI-labeled impinger samples yielded comparable estimates of bioaerosol concentrations when enumerated by microscopy or flow cytometry. The standard culture method underestimated bioaerosol concentrations by 2 orders of magnitude when compared to microscopy or flow cytometry. In the FISH method, aliquots of collection media were incubated with a probe universally complementary to eubacteria, a probe specific for several Pseudomonas species, and a probe complementary to eubacteria for detection of nonspecific binding. With these probes, FISH allowed quantitative identification of Pseudomonas aeruginosa and Escherichia coli bioaerosols in the exposure chamber without measurable nonspecific binding. Impinger samples from the swine barn demonstrated the efficacy of the FISH method for the identification of eubacteria in a complex organic dust. This work demonstrates the potential of emerging molecular techniques to complement traditional methods of bioaerosol exposure assessment. PMID:9097451

  7. Interphase fluorescence in situ hybridization signal detection by computing intensity variance along the optical axis

    NASA Astrophysics Data System (ADS)

    Li, Zheng; Zheng, Bin; Ren, Liqiang; Liu, Hong

    2014-02-01

    Fluorescence in situ Hybridization technology is a commonly used tool to detect chromosome aberrations, which are often pathologically significant. Since manual FISH analysis is a tedious and time-consuming procedure, reliable and robust automated image acquisition and analysis are in demand. Under high magnification objective lenses such as 60x and 100x, the depth of field will often be too small and the FISH probes may not always lie in the same focal plane. A statistical variance based automated FISH analysis method is developed in order to address this problem. On a stack of slices at consecutive image planes with a step size d, the statistical variance alone the z-axis is calculated to form a 2-D matrix. Since pixels shift dramatically to high intensity at FISH probe location, the probes will manifest high peak values in the matrix. A computer-aided detection scheme based on top-hat transform is applied to the matrix to detect FISH probe signals. This study demonstrates a simple and robust method for FISH probe detection as well as a way of 2- D representation of 3-D data.

  8. Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology

    PubMed Central

    2010-01-01

    Comet assay and micronucleus (MN) test are widely applied in genotoxicity testing and biomonitoring. While comet assay permits to measure direct DNA-strand breaking capacity of a tested agent MN test allows estimating the induced amount of chromosome and/or genome mutations. The potential of these two methods can be enhanced by the combination with fluorescence in situ hybridization (FISH) techniques. FISH plus comet assay allows the recognition of targets of DNA damage and repairing directly. FISH combined with MN test is able to characterize the occurrence of different chromosomes in MN and to identify potential chromosomal targets of mutagenic substances. Thus, combination of FISH with the comet assay or MN test proved to be promising techniques for evaluation of the distribution of DNA and chromosome damage in the entire genome of individual cells. FISH technique also permits to study comet and MN formation, necessary for correct application of these methods. This paper reviews the relevant literature on advantages and limitations of Comet-FISH and MN-FISH assays application in genetic toxicology. PMID:20840797

  9. In situ synthesis of TiO2/polyethylene terephthalate hybrid nanocomposites at low temperature

    NASA Astrophysics Data System (ADS)

    Peng, Xinyan; Ding, Enyong; Xue, Feng

    2012-06-01

    TiO2 nanoflowers were in situ grown on polyethylene terephthalate (PET) non-woven fabric by hydrolysis of TiCl4 in aqueous solution in the presence of nanocrystal cellulose grafted PET fabric (NCC-g-PET) at a low temperature of 70 °C. Nanocrystal cellulose (NCC) pre-grafted on PET fabric acted as hydrophilic substrate and morphology inducing agent to promote the nucleation and crystal growth of TiO2. Detailed information on the synthetic process was presented. The resulting samples were characterized using FE-SEM, EDS, ATR-IR, Raman microscopy, XRD and TG analysis. The photocatalytic activity of the samples was evaluated by the degradation of orange methyl under solar light. Characteristic results indicate that rutile TiO2 nanoflowers have grown abundantly on PET non-woven fabric, and the established hydrogen bonding strengthens the interfacial interaction between the inorganic particles and the polymeric substrates. The methyl orange decoloration test under natural solar light demonstrates that this TiO2/PET hybrid nanocomposites exhibit excellent self-cleaning performance which is expected to have a good potential for commercialization.

  10. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method.

    PubMed

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina; Cerca, Nuno

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6-99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  11. A whole-mount in situ hybridization method for microRNA detection in Caenorhabditis elegans.

    PubMed

    Andachi, Yoshiki; Kohara, Yuji

    2016-07-01

    Whole-mount in situ hybridization (WISH) is an outstanding method to decipher the spatiotemporal expression patterns of microRNAs (miRNAs) and provides important clues for elucidating their functions. The first WISH method for miRNA detection was developed in zebrafish. Although this method was quickly adapted for other vertebrates and fruit flies, WISH analysis has not been successfully used to detect miRNAs in Caenorhabditis elegans Here, we show a novel WISH method for miRNA detection in C. elegans Using this method, mir-1 miRNA was detected in the body-wall muscle where the expression and roles of mir-1 miRNA have been previously elucidated. Application of the method to let-7 family miRNAs, let-7, mir-48, mir-84, and mir-241, revealed their distinct but partially overlapping expression patterns, indicating that miRNAs sharing a short common sequence were distinguishably detected. In pash-1 mutants that were depleted of mature miRNAs, signals of mir-48 miRNA were greatly reduced, suggesting that mature miRNAs were detected by the method. These results demonstrate the validity of WISH to detect mature miRNAs in C. elegans. PMID:27154969

  12. Does polyomavirus infection interfere with bladder cancer fluorescence in situ hybridization?

    PubMed

    Hossain, Deloar; Hull, David; Kalantarpour, Fatemeh; Maitlen, Rebecca; Qian, Junqi; Bostwick, David G

    2014-03-01

    Urine cytology is a proven and widely used screening tool for the detection of urothelial carcinoma. However, morphologic features of polyomavirus infected cells, characterized by nuclear inclusions (decoy cells) are a known source of diagnostic confusion with malignancy. Fluorescence in situ hybridization (FISH) is now routinely used to support the cytological diagnosis of urothelial carcinoma and monitor for recurrence. We sought to determine whether polyomavirus infection could result in positive FISH results (aneuploidy). This study deals with retrospective study of 100 polyomavirus-infected urine samples from patients with no history of urothelial carcinoma or organ transplantation. All cases were stained with Papanicolaou and acid hematoxylin stain. One slide from each sample was de-stained and FISH was performed using chromosome enumeration probes 3, 7, 17, and locus-specific probe 9p21. Adequate cells for FISH analysis (25 cells) were present in 81 cases; 19 cases were insufficient due to loss of cells during de-staining and FISH preparation process. All polyomavirus-infected cells (decoy cells) exhibited a normal chromosome pattern. Four cases were FISH positive, but there were no positive decoy cells. Decoy cells did not exhibit aneuploidy by FISH. The presence of decoy cells does not exclude the possibility of concurrent urothelial carcinoma. Acid hematoxylin stain appeared to supplement the Papanicolou stain in identifying and confirming the presence of polyomavirus infection. PMID:24006232

  13. Three dimensional dual labelled DNA fluorescent in situ hybridization analysis in fixed tissue sections

    PubMed Central

    Kernohan, Kristin D.; Bérubé, Nathalie G.

    2014-01-01

    Emerging studies demonstrate that three-dimensional organization of chromatin in the nucleus plays a vital role in regulating the genome. DNA fluorescent in situ hybridization (FISH) is a common molecular technique used to visualize the location of DNA sequences. The vast majority of DNA FISH studies are conducted on cultured cells due to the technical difficulties encountered using fixed tissue sections. However, the use of cultured cells poses important limitations that could yield misleading results, making in vivo analysis a far superior approach. Here we present a protocol for multiplexed three dimensional DNA FISH in mouse brain sections, which is also applicable to other tissues. Paraffin-embedded tissues could be used but the embedding and preparation of the samples is time-consuming and often associated with poor antigenicity. To overcome this problem we:•developed a FISH technique using fixed, frozen cryosections;•provide specific instructions for tissue processing for proper fixation and freezing, including equilibration in sucrose gradients to maintain proper cellular structure;•include optimized permeabilization and washing steps to achieve specific signal and to limit background fluorescence in tissue sections. PMID:26150931

  14. Estimate of true incomplete exchanges using fluorescence in situ hybridization with telomere probes

    NASA Technical Reports Server (NTRS)

    Wu, H.; George, K.; Yang, T. C.

    1998-01-01

    PURPOSE: To study the frequency of true incomplete exchanges in radiation-induced chromosome aberrations. MATERIALS AND METHODS: Human lymphocytes were exposed to 2 Gy and 5 Gy of gamma-rays. Chromosome aberrations were studied using the fluorescence in situ hybridization (FISH) technique with whole chromosome-specific probes, together with human telomere probes. Chromosomes 2 and 4 were chosen in the present study. RESULTS: The percentage of incomplete exchanges was 27% when telomere signals were not considered. After excluding false incomplete exchanges identified by the telomere signals, the percentage of incomplete exchanges decreased to 11%. Since telomere signals appear on about 82% of the telomeres, the percentage of true incomplete exchanges should be even lower and was estimated to be 3%. This percentage was similar for chromosomes 2 and 4 and for doses of both 2 Gy and 5 Gy. CONCLUSIONS: The percentage of true incomplete exchanges is significantly lower in gamma-irradiated human lymphocytes than the frequencies reported in the literature.

  15. In Situ Growth of In2S3 Nanorods in Poly(3-Hexylthiophene) Hybrid Films

    NASA Astrophysics Data System (ADS)

    Cota-Leal, M.; Sotelo-Lerma, M.; Corona-Corona, I.; Quevedo-Lopez, M. A.

    2016-04-01

    A novel and efficient gas-liquid method for the in situ synthesis of In2S3 nanorods in a poly(3-hexylthiophene) (P3HT) matrix is demonstrated. The method involves a self-contained reaction between Na2S and HCl that produces H2S, which reacts with a P3HT/InCl3 solution resulting in hybrid P3HT/In2S3 films. The Na2S solution is regenerated for further use. The method yielded results in In2S3 nanoparticles and nanorods in a P3HT matrix, as observed by transmission electron microscopy. The In2S3 nanorods are 3 nm wide and ~30 nm long. The size of the nanorods is dependent on the P3HT concentration. The band gap (E g) of the resulting In2S3/P3HT is in the range of 2.97-3.71 eV, as measured by UV-visible spectroscopy (UV-Vis) Charge transfer in the In2S3/P3HT was demonstrated by the presence of quenching in the fluorescence spectra of the composite. Chemical composition was investigated by energy dispersive x-ray spectroscopy analysis, as well as x-ray photoelectron spectroscopy. Both techniques demonstrated the formation of In2S3.

  16. Identification of pathogens in mastitis milk samples with fluorescent in situ hybridization.

    PubMed

    Gey, Annerose; Werckenthin, Christiane; Poppert, Sven; Straubinger, Reinhard K

    2013-05-01

    Traditionally, the bacteriological examination of mastitis milk samples is performed by culture followed by biochemical tests on the cultured bacteria to allow identification of the causative pathogen. Depending on the species involved, this classic identification is time-consuming compared to other techniques such as fluorescent in situ hybridization (FISH), a culture-independent method that utilizes oligonucleotides (labeled with a fluorophore) that are specific to a string of target DNA/RNA. In the current study, the applicability of FISH was evaluated for the detection of mastitis pathogens directly in milk samples. To remove interfering lipids and proteins from mastitis milk samples prior to FISH, a previously published enzymatic treatment with savinase was evaluated. FISH was performed using oligonucleotides specific for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, and Trueperella (Arcanobacterium) pyogenes. The enzymatic pretreatment and the sensitivity of FISH were evaluated using spiked whole milk samples and mastitis milk samples with bacterial loads of less than 10(3) up to 10(8) colony-forming units (CFU)/ml. Bacteria were reliably detected in milk samples with bacterial numbers of 10(6) CFU/ml or higher. However, bacteria present in numbers below 10(6) CFU/ml were not detectable in all cases. The ability of FISH to identify mastitis-causing pathogens directly in milk samples, and therefore earlier than classical culture methods, can supplement the classic diagnostic procedures for mastitis milk samples. PMID:23632662

  17. Fluorescent In Situ Hybridization Allows Rapid Identification of Microorganisms in Blood Cultures

    PubMed Central

    Kempf, Volkhard A. J.; Trebesius, Karlheinz; Autenrieth, Ingo B.

    2000-01-01

    Using fluorescent in situ hybridization (FISH) with rRNA-targeted fluorescently labelled oligonucleotide probes, pathogens were rapidly detected and identified in positive blood culture bottles without cultivation and biotyping. In this study, 115 blood cultures with a positive growth index as determined by a continuous-reading automated blood culture system were examined by both conventional laboratory methods and FISH. For this purpose, oligonucleotide probes that allowed identification of approximately 95% of those pathogens typically associated with bacteremia were produced. The sensitivity and specificity of these probes were 100%. From all 115 blood cultures, microorganisms were grown after 1 day and identification to the family, genus, or species level was achieved after 1 to 3 days while 111 samples (96.5%) were similarly identified by FISH within 2.5 h. Staphylococci were identified in 62 of 62 samples, streptococci and enterococci were identified in 19 of 20 samples, gram-negative rods were identified in 28 of 30 samples, and fungi were identified in two of two samples. Thus, FISH is an appropriate method for identification of pathogens grown in blood cultures from septicemic patients. PMID:10655393

  18. Simple Adhesive-Tape-Based Sampling of Tomato Surfaces Combined with Rapid Fluorescence In Situ Hybridization for Salmonella Detection▿

    PubMed Central

    Bisha, Bledar; Brehm-Stecher, Byron F.

    2009-01-01

    A simple adhesive-tape-based method for sampling of tomato surfaces was combined with fluorescence in situ hybridization for rapid culture-independent detection of Salmonella strains. Tapes could also be placed face-down on selective agar for on-tape enrichment of captured Salmonella cells. Overlay of cell-charged tapes with small volumes of liquid enrichment media enabled subsequent detection of tape-captured Salmonella via flow cytometry. PMID:19124588

  19. Visualization of the mycelia of wood-rotting fungi by fluorescence in situ hybridization using a peptide nucleic acid probe.

    PubMed

    Nakada, Yuji; Nakaba, Satoshi; Matsunaga, Hiroshi; Funada, Ryo; Yoshida, Makoto

    2013-01-01

    White rot fungus, Phanerochaete chrysosporium, and brown rot fungus, Postia placenta, grown on agar plates, were visualized by fluorescence in situ hybridization (FISH) using a peptide nucleic acid (PNA) probe. Mycelia grown on wood chips were also clearly detected by PNA-FISH following blocking treatment. To the best of our knowledge, this is the first report on the visualization of fungi in wood by FISH. PMID:23391931

  20. Fetal t(5p;21q) misdiagnosed as monosomy 21: A plea for in situ hybridization studies

    SciTech Connect

    Gill, P.; Uhrich, S.; Cheng, E.; Disteche, C.

    1994-10-01

    We report a case of 45,XY,-5,-21,+der (5)t(5;21) (p13 or p14;q11.2 or q21) that was prenatally misdiagnosed as complete monosomy 21 and terminated at 24 weeks of gestation. Subsequent fluorescence in situ hybridization studies with a chromosome 21 painting probe documented the cryptic unbalanced translocation. 17 refs., 2 figs., 1 tab.

  1. Immuno-Electron Microscopy and Electron Microscopic In Situ Hybridization for Visualizing piRNA Biogenesis Bodies in Drosophila Ovaries.

    PubMed

    Shibata, Shinsuke; Murota, Yukiko; Nishimoto, Yoshinori; Yoshimura, Mana; Nagai, Toshihiro; Okano, Hideyuki; Siomi, Mikiko C

    2015-01-01

    Immuno-electron microscopy and electron microscopic in situ hybridization are powerful tools to identify the precise subcellular localization of specific proteins and RNAs at the ultramicroscopic level. Here we describe detailed procedures for how to detect the precise location of a specific target labeled with both fluorescence and gold particles. Although they have been developed for the analysis of Drosophila ovarian somatic cells, these techniques are suitable for a wide range of biological applications including human, primate, and rodent analysis. PMID:26324437

  2. Localization of the human OB gene (OBS) to chromosome 7q32 by fluorescence in situ hybridization

    SciTech Connect

    Geffroy, S.; Duban, B.; Martinville, B. de

    1995-08-10

    An important gene involved in the pathogenesis of obesity is the product of the human homologue of the murine obese gene (gene symbol OBS). Using fluorescence in situ hybridization (FISH), we have localized the human OB gene to human chromosome 7, specifically to region 7q32.1. The FISH data of human OBS provide a gene-associated marker for genetic mapping. 8 refs., 1 fig.

  3. An extended fluorescence in situ hybridization approach for the cytogenetic study of cholangiocarcinoma on endoscopic retrograde cholangiopancreatography brushing cytology preparations.

    PubMed

    Vasilieva, Larisa E; Papadhimitriou, Stefanos I; Alexopoulou, Alexandra; Pavlidis, Dimitris; Kostopoulos, Ioannis; Georgiakaki, Maria; Xinopoulos, Dimitrios; Romanos, Andreas; Dourakis, Spyridon P

    2013-10-01

    The cytological diagnosis of cholangiocarcinoma has been significantly aided by applying a 4-probe fluorescence in situ hybridization system on endoscopic retrograde cholangiopancreatography brushing smears, aiming mainly at the detection of hyperdiploidy. However, this approach adds little to our understanding of the genetic background of the disease. With the prospect of obtaining additional data on chromosomal aberrations, we have extended the fluorescence in situ hybridization study, with the application of 4 independent 2-probe systems in 35 patients with documented cholangiocarcinoma. Fluorescence in situ hybridization assays were performed on endoscopic retrograde cholangiopancreatography brushing smears, with probes for the 7q31, 11q13 (CCND1), 17p53 (TP53), and 9p21 (INK4 locus) bands, together with the respective centromeric probe. Hyperdiploidy, involving at least 2 of the 4 chromosomes targeted, was found in 31 patients. 17p13 deletion was detected in 3, and 9p21 deletion, in 5 of the hyperdiploid cases, with the 2 aberrations concurrent in 1. CCND1 amplification was found in 1 case as the sole abnormality and in another together with hyperdiploidy, but in apparently unrelated clones. This work indicates that interphase fluorescence in situ hybridization is a practical and useful tool for the cytogenetic study of cholangiocarcinoma on endoscopic retrograde cholangiopancreatography brushing smears, which is often the only available tissue specimen of the tumor. Apart from hyperdiploidy, it provides additional data on the genetic profile of cholangiocarcinoma, especially regarding structural chromosomal aberrations and clonal diversity. This line of investigation may prove useful in the delineation of oncogenesis and the interpretation of the diverse clinical features of the disease. PMID:23845469

  4. Miniaturised 'lab-on-a-chip' nitrate analyser applied to high resolution in situ analysis of glacial meltwater

    NASA Astrophysics Data System (ADS)

    Beaton, A.; Mowlem, M.; Wadham, J. L.

    2013-12-01

    In situ chemical measurements of glacial meltwater can provide high temporal and spatial resolution data that allow us to infer biogeochemical processes and calculate export from glacial systems. Despite this, in situ measurements of single chemical parameters in glacial meltwater have so far largely been restricted to pH and dissolved oxygen. The lack of high performance ruggedized in situ sensors for other analytes means that the laboratory-based analysis of manually collected samples is still routine. Microfluidics (through lab-on-a-chip technology) permits the miniaturisation of established chemical analysis techniques so that they can be performed in situ. The advantages of decreased size and low power and reagent consumption make these systems suitable for deployment in extreme and inaccessible environments where regular manual sample collection is logistically difficult. We present data from a novel stand-alone microfluidic wet chemical nitrate analyser that has been deployed to monitor a proglacial meltwater river draining from the Greenland ice sheet. By performing a measurement every 20 minutes, the analyser was able to reveal diurnal fluctuations and short term trends in nitrate concentrations that would not discernible using standard daily sampling. High resolution in situ measurements such as these can allow a more accurate determination of nutrient export fluxes from glacial systems into the polar oceans, and allow enhanced interpretation of water quality datasets. Steps have been taken to ruggedize the system so that it can survive the freeze-thaw conditions, dilute concentrations and high sediment loads that can be associated with cryospheric environments. The system is small, has low power consumption and detects nitrate and nitrite with a limit of detection (LOD) of 0.025 μM, which is sufficient for low nutrient glacial environments. On-going work looks to deploy similar nutrient analysers more widely, not only in glacial systems, but also in

  5. A comparison of two methods for colorimetric in situ hybridization using paraffin-embedded tissue sections and digoxigenin-labeled hybridization probes.

    PubMed

    Marcino, Joe

    2013-06-01

    Two methods for colorimetric in situ DNA probe hybridization (CISH) assays on paraffin-embedded tissue sections were compared. The heated method used heat (90-100°C) to denature DNA in the sample prior to probe hybridization, while the unheated method used a standard hybridization temperature of 42°C. Both procedures were tested on tissue samples that harbored the mollusk protozoan pathogens Perkinsus marinus, P. chesapeaki, or Haplosporidium nelsoni, the protozoan and bacterial fish pathogens Myxobolus cerebralis (myxosporidean) or Renibacterium salmoninarum (bacterial), or the crab viral pathogen Callinectes sapidus reovirus. Samples were fixed in either formalin or Davidson's fixative and embedded in paraffin for histological examination. The heated method is labor intensive and highly prone to human error, while the unheated method is less labor intensive and can be completed in a shorter period of time. Both methods yielded similar hybridization results. The use of complex and expensive prehybridization buffers did not improve the performances of the tested CISH assays. Prehybridization heat denaturation of DNA in assayed samples increased both assay duration and loss of samples but did not improve hybridization signals. PMID:23697605

  6. Babesia gibsoni: detection in blood smears and formalin-fixed, paraffin-embedded tissues using deoxyribonucleic acid in situ hybridization analysis.

    PubMed

    Yamasaki, Masahiro; Kobayashi, Yusuke; Nakamura, Kensuke; Sasaki, Noboru; Murakami, Masahiro; Rajapakshage, Bandula Kumara Wickramasekara; Ohta, Hiroshi; Yamato, Osamu; Maede, Yoshimitsu; Takiguchi, Mitsuyoshi

    2011-01-01

    In this study, we attempted to detect Babesia gibsoni in blood smears and formalin-fixed, paraffin-embedded tissues obtained from B. gibsoni-infected dogs using in situ hybridization. Using a digoxigenin-conjugated deoxyribonucleic acid (DNA) probe, both intraerythrocytic and exoerythrocytic parasites in the culture could be specifically stained in blood smears fixed with 4% phosphate-buffered paraformaldehyde. This indicated that genomic DNA extracted from the parasites could be detected using in situ hybridization. Moreover, the parasite could be specifically stained in paraffin-embedded spleen, lymph node, and kidney sections using in situ hybridization. Infected erythrocytes in blood vessels in the spleen and kidney, hemosiderin-laden macrophages in the spleen, and phagocytized erythrocytes, which seemed to be infected with the parasites, in lymph nodes were also specifically stained. This suggests that in situ hybridization can be utilized to investigate both the life cycle of B. gibsoni and the pathological condition of canine babesiosis. PMID:20637756

  7. Vaginal micropapillary lesions are not related to human papillomavirus infection: in situ hybridization and polymerase chain reaction detection techniques.

    PubMed

    Garzetti, G G; Ciavattini, A; Goteri, G; Menzo, S; De Nictolis, M; Clementi, M; Brugia, M; Romanini, C

    1994-01-01

    The objective of this study was to assess the human papillomavirus DNA presence in vaginal papillary lesions, with particular regard to micropapillomatosis to better define their clinical significance. Prospective study: the study population was composed of 62 women who were recruited consecutively from the Colposcopy Centre of the Ancona University, Department of Obstetrics and Gynecology, on the grounds of vaginal papillomatosis or/and typical acuminata warts. Biopsies for routine histology, and for human papillomavirus (HPV) DNA detection by means of in situ hybridization and polymerase chain reaction (PCR) were taken from the papillary lesions and from 24 healthy women, who were selected as controls. Macroscopically, vaginal micropapillomatosis was ascertained in 51 cases (82.3%), while in 11 cases (17.7%) the colposcopic diagnosis was condyloma acuminatum. During in situ hybridization, HPV DNA positivity was observed in 8 (9.4%) out of 85 samples of squamous papillae and in 11 (64.7%) out of 17 samples of condylomata; in control specimens, HPV DNA was detected in 2 (8.3%) out of 24 bioptic samples. The correspondence between in situ hybridization and PCR was 96.1%, with 17.4% more diagnosis obtained by PCR. Vaginal micropapillomatosis may be regarded as a variation in the normal anatomy of the lower genital tract without any significant relationship with HPV infection, and as a lesion easily distinguishable from condylomata acuminata by clinical examination alone. PMID:7959342

  8. Detection and quantification of Epstein-Barr virus EBER1 in EBV-infected cells by fluorescent in situ hybridization and flow cytometry

    NASA Technical Reports Server (NTRS)

    Stowe, R. P.; Cubbage, M. L.; Sams, C. F.; Pierson, D. L.; Barrett, A. D.

    1998-01-01

    A rapid and highly sensitive fluorescent in situ hybridization (FISH) assay was developed to detect Epstein Barr virus (EBV)-infected cells in peripheral blood. Multiple fluorescein-labeled antisense oligonucleotide probes were designed to hybridize to the EBER1 transcript, which is highly expressed in latently infected cells. After a rapid (30 min) hybridization, the cells were analyzed by flow cytometry. EBER1 was detected in several positive control cell lines that have variable numbers of EBV genome copies. No EBER1 was detected in two known EBV-negative cell lines. Northern blot analyses confirmed the presence and quantity of EBER1 transcripts in each cell line. This method was used to quantify the number of EBV-infected cells in peripheral blood from a patient with chronic mononucleosis. These results indicate that EBV-infected cells can be detected at the single cell level, and that this assay can be used to quantify the number of EBV-infected cells in clinical samples.

  9. Synthesis and cytological analyses of hybrids between hexaploid wheat, with and without Ph1, and diploid wheatgrass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The usefulness of wide hybridization in genetic enhancement of crop plants is well documented. Diploid wheatgrass is a source of several desirable traits including Fusarium head blight resistance. The objective of this study was to report on the synthesis and cytological analyses of wheat × wheatg...

  10. Novel Hybrid Monte Carlo/Deterministic Technique for Shutdown Dose Rate Analyses of Fusion Energy Systems

    SciTech Connect

    Ibrahim, Ahmad M; Peplow, Douglas E.; Peterson, Joshua L; Grove, Robert E

    2013-01-01

    The rigorous 2-step (R2S) method uses three-dimensional Monte Carlo transport simulations to calculate the shutdown dose rate (SDDR) in fusion reactors. Accurate full-scale R2S calculations are impractical in fusion reactors because they require calculating space- and energy-dependent neutron fluxes everywhere inside the reactor. The use of global Monte Carlo variance reduction techniques was suggested for accelerating the neutron transport calculation of the R2S method. The prohibitive computational costs of these approaches, which increase with the problem size and amount of shielding materials, inhibit their use in the accurate full-scale neutronics analyses of fusion reactors. This paper describes a novel hybrid Monte Carlo/deterministic technique that uses the Consistent Adjoint Driven Importance Sampling (CADIS) methodology but focuses on multi-step shielding calculations. The Multi-Step CADIS (MS-CADIS) method speeds up the Monte Carlo neutron calculation of the R2S method using an importance function that represents the importance of the neutrons to the final SDDR. Using a simplified example, preliminarily results showed that the use of MS-CADIS enhanced the efficiency of the neutron Monte Carlo simulation of an SDDR calculation by a factor of 550 compared to standard global variance reduction techniques, and that the increase over analog Monte Carlo is higher than 10,000.