scholarly journals Sensitive detection of rat gastrin mRNA by in situ hybridization with chemically biotinylated oligodeoxynucleotides: validation, quantitation, and double-staining studies.

1993 ◽  
Vol 41 (2) ◽  
pp. 157-163 ◽  
Author(s):  
L I Larsson ◽  
D M Hougaard
Keyword(s):  
In Situ Hybridization ◽  
Detection System ◽  
Northern Blotting ◽  
Model Systems ◽  
Double Staining ◽  
Adult Rats ◽  
Gastrin Cells ◽  
Human Gastrin ◽  
Almost All

Chemically biotin-labeled oligonucleotides form attractive reagents, as large quantities of stable and well-defined probes can easily be produced. Their usefulness for in situ hybridization was tested using rat gastrin cells as a model. Two probes recognizing two different regions of rat gastrin mRNA were synthesized and produced specific and equally strong hybridization signals. A probe complementary to human gastrin mRNA, but with mismatches to the rat gastrin mRNA sequence, failed to reveal rat gastrin cells under the stringency conditions used. Northern blotting revealed that the rat gastrin mRNA probes reacted exclusively with the appropriately sized (approximately 650 bases) mRNA. Model systems demonstrated that the hybridization signal, as revealed by alkaline phosphatase-based detection, varied linearly with the 10logarithm of target concentration and also showed that a new detection system was much more sensitive than previously used systems. In agreement with previous biochemical data, image analysis showed that starvation of rats led to a progressive decrease in cell staining intensities and cell numbers. Double staining for rat gastrin mRNA and gastrin immunoreactivity showed that in adult rats almost all gastrin cells expressed both mRNA and protein. Similar studies on developing rat gastrin cells revealed discrepancies between gastrin mRNA and gastrin-immunoreactive cells during the first week of newborn life. Subsequently, expression of mRNA and protein in the cells became gradually more concordant.

scholarly journals Comparative molecular cytogenetics in Melipona Illiger species (Hymenoptera, Apidae)

Sociobiology ◽  
2018 ◽  
Vol 65 (4) ◽  
pp. 696 ◽  
Author(s):  
Vanderly Andrade-Souza ◽  
Olivia Maria Pereira Duarte ◽  
Cinthia Caroline Cardoso Martins ◽  
Igor Silva Santos ◽  
Márcio Gilberto Cardoso Costa ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Chromosome Number ◽  
Fluorescent In Situ Hybridization ◽  
Molecular Cytogenetics ◽  
Fluorochrome Staining ◽  
Rdna Sites ◽  
Cytogenetic Studies ◽  
Melipona Species ◽  
Almost All

Cytogenetic studies in Melipona are scarce with only 24 species analyzed cytogenetically. Of these, six species had the rDNA sites physically mapped and characterized by Fluorescent in situ Hybridization (fish). The aim of this study was to perform karyotype analyzes on Melipona species from different regions of Brazil, with a greater sampling representative of the Amazonian fauna and using conventional, fluorochrome staining and FISH with heterologous rDNA probes. The predominant chromosome number was 2n = 18, however, the subspecies M. seminigra abunensis and M. s. pernigra showed 2n = 22 chromosomes. The karyotypes were symmetrical, however M. bicolor, M. quadrifasciata, M. flavolineata, M. fuscopilosa, M. nebulosa presented the first pair heteromorphic in length. CMA3+ blocks also exhibited heteromorphism of size and in almost all cases coincided with rDNA sites, except for M. crinita and M. nebulosa, which presented additional non-coincident CMA3+ blocks. The CMA/ rDNA sites were terminal and interstitial in species with high heterochromatic content, and pericentromeric in those species with low heterochromatic content. In addition to pointing out cytogenetic features of cytotaxonomic importance, the reorganization of the genome in Melipona is discussed.

scholarly journals Fluorescence In Situ Hybridization (FISH) Analysis of the Locations of the Oligonucleotides 5S rDNA, (AGGGTTT)3, and (TTG)6 in Three Genera of Oleaceae and Their Phylogenetic Framework

Genes ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 375 ◽  
Author(s):  
Xiaomei Luo ◽  
Juncheng Liu
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
5S Rdna ◽  
Fish Analysis ◽  
Cytogenetic Map ◽  
Ligustrum Lucidum ◽  
Starting Point ◽  
Central Regions ◽  
Almost All

We report the cytogenetic map for a collection of species in the Oleaceae, and test similarities among the karyotypes relative to their known species phylogeny. The oligonucleotides 5S ribosomal DNA (rDNA), (AGGGTTT)3, and (TTG)6 were used as fluorescence in situ hybridization (FISH) probes to locate the corresponding chromosomes in three Oleaceae genera: Fraxinus pennsylvanica, Syringa oblata, Ligustrum lucidum, and Ligustrum × vicaryi. Forty-six small chromosomes were identified in four species. (AGGGTTT)3 signals were observed on almost all chromosome ends of four species, but (AGGGTTT)3 played no role in distinguishing the chromosomes but displayed intact chromosomes and could thus be used as a guide for finding chromosome counts. (TTG)6 and 5S rDNA signals discerned several chromosomes located at subterminal or central regions. Based on the similarity of the signal pattern (mainly in number and location and less in intensity) of the four species, the variations in the 5S rDNA and (TTG)6 distribution can be ordered as L. lucidum < L. × vicaryi < F. pennsylvanica < S. oblata. Variations have observed in the three genera. The molecular cytogenetic data presented here might serve as a starting point for further larger-scale elucidation of the structure of the Oleaceae genome, and comparison with the known phylogeny of Oleaceae family.

scholarly journals Expression of the c-fos protooncogene by human and murine erythroblasts

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 947-951 ◽  
Author(s):  
JF Caubet ◽  
MT Mitjavila ◽  
A Dubart ◽  
D Roten ◽  
SC Weil ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Northern Blotting ◽  
Hematopoietic Tissue ◽  
Double Labeling ◽  
Murine Bone Marrow ◽  
Marrow Cells

Abstract The expression of the c-fos protooncogene was investigated by in situ hybridization in normal murine bone marrow cells. A strong signal was found in murine marrow cells having the morphologic features of erythroblasts. This result was confirmed in human marrow cells using a double labeling technique (in situ hybridization and immunocytochemistry). A majority (70%) of the cells expressing c-fos mRNA were glycophorin A-positive. In contrast, granulocytic precursors (CD 15-positive) or monocytes and their precursors (CD 14-positive cells) did not significantly hybridize with the c-fos probe. In addition, c-fos mRNA (2.2Kb) was detected by Northern blotting in RNA extracted from homogeneous populations of erythroblasts obtained by immune panning from fetal liver and from adult blood BFU-E-derived colonies. Fos protein was also detected in erythroblasts by immunofluorescence. The high level of c-fos mRNA previously found in hematopoietic tissue should therefore be related to the transcription of the c-fos gene during terminal erythroid differentiation.

scholarly journals Enhanced chemiluminescence: a high-sensitivity detection system for in situ hybridization and immunohistochemistry.

1993 ◽  
Vol 41 (11) ◽  
pp. 1591-1597 ◽  
Author(s):  
P Lorimier ◽  
L Lamarcq ◽  
F Labat-Moleur ◽  
C Guillermet ◽  
R Bethier ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Detection System ◽  
Photon Counting ◽  
Protein Detection ◽  
High Sensitivity ◽  
Papilloma Virus ◽  
Enhanced Chemiluminescence ◽  
Discriminatory Analysis ◽  
High Sensitivity Detection

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

Expression of a Bet v 1 homologue gene encoding a PR 10 protein in birch roots: induction by auxin and localization of the transcripts by in situ hybridization

2001 ◽  
Vol 28 (1) ◽  
pp. 57 ◽  
Author(s):  
Pascal Poupard ◽  
Nicole Brunel ◽  
Nathalie Leduc ◽  
Jean-Daniel Viémont ◽  
Désiré-Georges Strullu ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Abiotic Factors ◽  
Lateral Roots ◽  
Northern Blotting ◽  
Root Tip ◽  
Gene Encoding ◽  
High Accumulation ◽  
Bet V 1 ◽  
Polymerase Chain

In birch roots (Betula pendula Roth), two members of the Bet v 1 gene family which encode PR 10 proteins have previously been characterized. One of these members, named Bet v 1-sc1, is significantly induced in response to biotic or abiotic factors. We have analysed the expression of Bet v 1-sc1 in birch roots treated either with 1 M indole-3-acetic acid (IAA) or 1 M kinetin using reverse transcription–polymerase chain reaction (RT–PCR), northern blotting and competitive PCR. High accumulation of the Bet v 1-sc1 transcripts was recorded only after auxin application, while kinetin had no effect. By in situ hybridization, we have investigated the localization of Bet v 1-sc1 mRNA in birch roots after induction of the gene by root treatment with 1 M IAA. Using root tip sections, we showed that Bet v 1-sc1 is significantly expressed in the apical meristem and the procambium. In sections taken in the zone producing lateral roots, the presence of Bet v 1-sc1 was found at sites of emerging secondary root primordia. This first report of localization of Bet v 1-sc1 expression suggests that this gene could be involved in the processes leading to lateral root initiation.

scholarly journals EnVision+, a New Dextran Polymer-based Signal Enhancement Technique for In Situ Hybridization (ISH)

2001 ◽  
Vol 49 (9) ◽  
pp. 1067-1071 ◽  
Author(s):  
Klaus Hermann Wiedorn ◽  
Torsten Goldmann ◽  
Christof Henne ◽  
Heike Kühl ◽  
Ekkehard Vollmer
Keyword(s):  
In Situ Hybridization ◽  
Signal Amplification ◽  
Detection System ◽  
Lower Sensitivity ◽  
The Novel ◽  
Visualization System ◽  
Integrated Optical ◽  
Assay Time ◽  
First Time

Seventy paraffin-embedded cervical biopsy specimens and condylomata were tested for the presence of human papillomavirus (HPV) by conventional in situ hybridization (ISH) and ISH with subsequent signal amplification. Signal amplification was performed either by a commercial biotinyl-tyramide-based detection system [GenPoint (GP)] or by the novel two-layer dextran polymer visualization system EnVision+ (EV), in which both EV–horseradish peroxidase (EV–HRP) and EV–alkaline phosphatase (EV–AP) were applied. We could demonstrate for the first time, that EV in combination with preceding ISH results in a considerable increase in signal intensity and sensitivity without loss of specificity compared to conventional ISH. Compared to GP, EV revealed a somewhat lower sensitivity, as measured by determination of the integrated optical density (IOD) of the positively stained cells. However, EV is easier to perform, requires a shorter assay time, and does not raise the background problems that may be encountered with biotinyl–tyramide-based amplification systems. (J Histochem Cytochem 49:1067–1071, 2001)

An Improved Nonfluorescent Detection System for in Situ Hybridization in Plants

2000 ◽  
Vol 75 (2) ◽  
pp. 49-53 ◽  
Author(s):  
Hanna Weiss ◽  
Pawel Pasierbek ◽  
Jolanta Maluszynska
Keyword(s):  
In Situ Hybridization ◽  
Detection System

scholarly journals Combined beta-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situ hybridization.

1990 ◽  
Vol 38 (3) ◽  
pp. 325-329 ◽  
Author(s):  
W van den Brink ◽  
C van der Loos ◽  
H Volkers ◽  
R Lauwen ◽  
F van den Berg ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Silver Staining ◽  
Staining Method ◽  
Double Staining ◽  
Reaction Products ◽  
Silver Enhancement ◽  
Silver Staining Method ◽  
Double Staining Method ◽  
Beta Galactosidase

A combination of beta-galactosidase enzyme and the immunogold/silver staining method was studied for evaluation of double-staining experiments. Applications are shown for immunohistochemical double staining using two monoclonal antibodies and for combined immunohistochemistry and DNA in situ hybridization in one tissue section. The following advantages for the present double-staining method were evaluated: superior sensitivity of the immunogold/silver staining method for at least one epitope, which also allows detection of biotinylated DNA probes. The structure of the indolyl precipitate after revelation of beta-galactosidase activity did not show a concealing effect during a sequential double-staining method, as compared with the visualization of peroxidase with diaminobenzidine. These factors, and the sharply contrasting colored reaction products of beta-galactosidase (blue-green) and the immunogold/silver staining method including silver enhancement (brown-black), allow clear distinction of mixed-stained cell constituents.

Perforin gene expression in stimulated human peripheral blood T cells studied by in situ hybridization and Northern blotting analysis

1992 ◽  
Vol 33 (1) ◽  
pp. 79-85
Author(s):  
Chau-Ching Liu ◽  
Shahin Rafii ◽  
Hirotaka Koizumi ◽  
Angela Granelli-Piperno ◽  
John Ding-E Young
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
In Situ Hybridization ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Northern Blotting ◽  
Blotting Analysis
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