scholarly journals Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin frozen sections vs post-embedding.

1992 ◽  
Vol 40 (7) ◽  
pp. 979-986 ◽  
Author(s):  
D Le Guellec ◽  
A Trembleau ◽  
C Pechoux ◽  
F Gossard ◽  
G Morel
Keyword(s):  
In Situ Hybridization ◽  
Detection System ◽  
Frozen Section ◽  
Ultrastructural Level ◽  
Pituitary Cells ◽  
Frozen Sections ◽  
Thin Sections ◽  
Section Method ◽  
Enzymatic Detection

In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultra-thin frozen sections, or on ultra-thin sections of tissues embedded in hydrophilic resin such as Lowicryl. With the purpose of comparing the sensitivity, resolution, and ultrastructural preservation of these three methods, we examined the expression of the growth hormone (GH) gene in anterior pituitary cells by in situ hybridization at the ultrastructural level, using a synthetic oligonucleotide complementary to the codons of the mRNA from Gln 45 to Ser 54 labeled at the 3' end of biotin-21dUTP. All these methods gave similar results: mRNA was located on the lamellar endoplasmic reticulum of somatotrophs. The pre-embedding method gave the best ultrastructural preservation, with low resolution with the enzymatic detection system and an intermediate sensitivity. A probe concentration of 10 pmol/ml was sufficient to obtain a signal. With this method gold particles could not be used without pre-treatment. The frozen section method gave the best sensitivity (a signal was observed with 4 pmol/ml of probe) but the lowest ultrastructural preservation. On ultra-thin Lowicryl sections, resolution was as high as with the frozen-section method, ultrastructural conservation was intermediate, and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation.

Enzymatic detection systems for non-isotopic in situ hybridization using biotinylated cDNA probes

1995 ◽  
Vol 27 (4) ◽  
pp. 280-290 ◽  
Author(s):  
Andreas Trabandt ◽  
Renate E. Gay ◽  
Vikas P. Sukhatme ◽  
Steffen Gay
Keyword(s):  
In Situ Hybridization ◽  
Detection Systems ◽  
Enzymatic Detection

scholarly journals Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach.

1991 ◽  
Vol 39 (11) ◽  
pp. 1495-1506 ◽  
Author(s):  
P M Motte ◽  
R Loppes ◽  
M Menager ◽  
R Deltour
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
Spatial Organization ◽  
Higher Plants ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Thin Sections ◽  
Cytoplasmic Dna ◽  
Immunocytochemical Techniques

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.

scholarly journals Immunodetection of RNA on ultra-thin sections incubated with polyadenylate nucleotidyl transferase.

1993 ◽  
Vol 41 (5) ◽  
pp. 657-665 ◽  
Author(s):  
M Thiry
Keyword(s):  
Divalent Cations ◽  
Ultrastructural Level ◽  
Ehrlich Tumor ◽  
Precise Location ◽  
Great Precision ◽  
Thin Sections ◽  
Nucleotidyl Transferase ◽  
Ehrlich Tumor Cells ◽  
Labeling Pattern

A new method is described for locating RNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing polyadenylate nucleotidyl transferase (PnT) and biotinylated ATP. The labeled nucleotides bound to RNA at the surface of the ultra-thin sections were than visualized by an indirect immunogold labeling technique. The resulting labeling pattern was dependent on the presence of divalent cations in the PnT medium. The method revealed with great precision the specific RNA-containing structures within Ehrlich tumor cells. The method is applicable to Epon sections. However, the labeling intensity varies according to the fixation used. Best results were obtained on acetylated cell sections. The method can be combined with EDTA regressive staining. The in situ PnT method provides a very useful tool for pinpointing the precise location of RNA within biological material at the ultrastructural level.

Genomic in situ hybridization to sectioned nuclei shows chromosome domains in grass hybrids

1990 ◽  
Vol 95 (3) ◽  
pp. 335-341
Author(s):  
A.R. Leitch ◽  
W. Mosgoller ◽  
T. Schwarzacher ◽  
M.D. Bennett ◽  
J.S. Heslop-Harrison
Keyword(s):  
In Situ Hybridization ◽  
Genomic Dna ◽  
Microscope Level ◽  
Hordeum Chilense ◽  
Root Tip ◽  
Interphase Nuclei ◽  
Light Microscope Level ◽  
Thin Sections ◽  
Electron Microscopes

In situ hybridization using biotinylated total genomic DNA and avidin detection systems was adapted for examination of thin-sectioned plant material in the light and electron microscopes. Root tip material was preserved prior to sectioning, so that the in vivo disposition of the chromatin was maintained. Use of total genomic DNA from Secale africanum as a probe enabled the chromatin from the two parental genomes in the grass hybrid Hordeum chilense × S. africanum to be distinguished. The biotinylated probe preferentially labelled the chromosomes of S. africanum origin. DNA-DNA hybrids were visualized at the light-microscope level by Texas Red fluorescence and at the electron-microscope level by the enzymic precipitation of DAB (diaminobenzidine) or by colloidal gold particles. The use of thin sections allowed the location of probe hybridization to be established unequivocally in both metaphase and interphase nuclei. Analysis of interphase nuclei showed that chromatin originating from the two parental genomes did not intermix but occupied distinct domains.

Urocortin: a mechanism for the sustained activation of the HPA axis in the late-gestation ovine fetus?

2002 ◽  
Vol 283 (1) ◽  
pp. E165-E171 ◽  
Author(s):  
Alison C. Holloway ◽  
David C. Howe ◽  
Gabriel Chan ◽  
Vicki L. Clifton ◽  
Roger Smith ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Late Gestation ◽  
Pituitary Cells ◽  
Fetal Sheep ◽  
Antisense Probe ◽  
Pomc Mrna ◽  
Ovine Fetus ◽  
Ovine Fetal ◽  
Sustained Activation

We hypothesized that urocortin might be produced in the pituitary of the late-gestation ovine fetus in a manner that could contribute to the regulation of ACTH output. We used in situ hybridization and immunohistochemistry to identify urocortin mRNA and protein in late-gestation fetal pituitary tissue. Levels of urocortin mRNA rose during late gestation and were associated temporally with rising concentrations of pituitary proopiomelanocortin (POMC) mRNA. Urocortin was localized both to cells expressing ACTH and to non-ACTH cells by use of dual immunofluorescence histochemistry. Transfection of pituitary cultures with urocortin antisense probe reduced ACTH output, whereas added urocortin stimulated ACTH output from cultured pituitary cells. Cortisol infusion for 96 h in chronically catheterized late-gestation fetal sheep significantly stimulated levels of pituitary urocortin mRNA. We conclude that urocortin is expressed in the ovine fetal pituitary and localizes with, and can stimulate output of, ACTH. Regulation of urocortin by cortisol suggests a mechanism to override negative feedback and sustain feedforward of fetal hypothalamic-pituitary-adrenal function, leading to birth.

scholarly journals Differential Regulation of Proopiomelanocortin and Pituitary-Restricted Transcription Factor (TPIT), a New Marker of Normal and Adenomatous Human Corticotrophs

2003 ◽  
Vol 88 (7) ◽  
pp. 3050-3056 ◽  
Author(s):  
Sophie Vallette-Kasic ◽  
Dominique Figarella-Branger ◽  
Michel Grino ◽  
Anne-Marie Pulichino ◽  
Henry Dufour ◽  
...  
Keyword(s):  
Transcription Factor ◽  
In Situ Hybridization ◽  
Pituitary Adenomas ◽  
Experimental Models ◽  
Pituitary Cells ◽  
Human Pituitary ◽  
T Box ◽  
Normal Human ◽  
Human Anterior Pituitary

Since the identification of the pituitary-restricted transcription factor Tpit, a novel T-box factor that is only present in mouse in the two pituitary proopiomelanocortin (POMC)-expressing lineages, no information was available on its pattern of expression in human pituitary. We investigated by immunohistochemistry and in situ hybridization the expression of TPIT in normal human anterior pituitary tissue and in several types of human pituitary adenomas (n = 52). TPIT expression was restricted to the nucleus of normal or adenomatous human corticotroph cells. No specific TPIT immunostaining was detectable in all prolactin (PRL)-, GH-, or gonadotropin-secreting adenomas. In situ hybridization studies demonstrated that TPIT transcripts were coexpressed with POMC mRNA in both secreting and silent corticotroph adenomas, and in normal corticotrophs, whereas TPIT mRNA was not detectable in other types of pituitary adenomas. Unlike POMC, TPIT was not up-regulated by adrenalectomy in rats and did not seem down-regulated in the normal pituitary adjacent to human corticotroph microadenomas. TPIT is the only currently known transcription factor selectively expressed in human normal and adenomatous corticotrophs. In human and experimental models, TPIT and its target gene POMC were thus differentially regulated by glucocorticoids. Moreover, TPIT represents a new marker of POMC-expressing pituitary cells.

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.

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)

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