In Situ Hybridization for RNA: Nonradioactive Probe: ss cDNA Probe

2000 ◽  
pp. 139-152
Author(s):  
Yoshio Kanemitsu ◽  
Takehiko Koji
Keyword(s):  
In Situ Hybridization ◽  
Cdna Probe

scholarly journals In situ detection of vitamin D-induced calcium-binding protein (9-kDa CaBP) messenger RNA in rat duodenum.

1986 ◽  
Vol 34 (2) ◽  
pp. 277-280 ◽  
Author(s):  
M Warembourg ◽  
O Tranchant ◽  
C Perret ◽  
C Desplan ◽  
M Thomasset
Keyword(s):  
Vitamin D ◽  
In Situ Hybridization ◽  
Calcium Binding ◽  
Binding Protein ◽  
Cdna Probe ◽  
Calcium Binding Protein ◽  
Pbr322 Dna ◽  
Rat Duodenum ◽  
Columnar Cells

We have previously described the molecular cloning of a cDNA fragment synthesized from rat duodenal mRNA coding for a 9000-dalton vitamin D-induced calcium-binding protein (9-kDa CaBP) (3). We now report the use of this cloned cDNA to study the cytological distribution of 9-kDa CaBP mRNA in rat duodenum by in situ hybridization. Tissue sections, fixed in ethanol:acetic acid, were hybridized to the 3H-cDNA probe and processed for autoradiography. The specificity of the CaBP mRNA-DNA hybrid formation was checked using 3H-labeled plasmid pBR322 DNA as a control probe. 9k-Da CaBP mRNA, visualized by silver grains, was found only in the absorptive epithelial cells, and the concentration was greater in the cells at the villous tips than in those of the crypts. The 9k-Da CaBP mRNA was observed mainly in the cytoplasm of the columnar cells and less frequently in the nucleus. Labeling was not seen in the brush border and goblet cells. The submucosa, with Brunner's glands and muscularis, also showed no specific 9-kDa CaBP mRNA concentration. This demonstration of 9-kDa CaBP gene activity in the columnar cells of the rat duodenum illustrates the usefulness of in situ hybridization for characterization of specific cells involved in the expression of 1,25(OH)2 D3 activity.

Localization of the sites of synthesis and action of prolactin by immunocytochemistry and in-situ hybridization within the human utero-placental unit

1991 ◽  
Vol 7 (3) ◽  
pp. 241-247 ◽  
Author(s):  
W.-X. Wu ◽  
J. Brooks ◽  
M. R. Millar ◽  
W. L. Ledger ◽  
P. T. K. Saunders ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Culture Medium ◽  
Cdna Probe ◽  
Specific Hybridization ◽  
Decidual Cells ◽  
Its Gene ◽  
First Time ◽  
Silver Grains ◽  
Prolactin Mrna

ABSTRACT While the fetal pituitary synthesizes and releases prolactin, it is also produced within the utero-placental unit during pregnancy in women and has been localized in the amnion, chorion and decidua. However, it is not clear whether prolactin is synthesized within all these non-fetal pituitary tissues. We have investigated prolactin production and its gene expression using tissue culture, immunocytochemistry and in-situ hybridization techniques. Prolactin was immunolocalized not only in the decidua but also in amnion and trophoblast cells. In contrast, the in-situ hybridization results showed that silver grains, formed by specific hybridization of a prolactin cDNA probe to prolactin mRNA, were confined to decidual cells of early and term pregnancy. The results from tissue cultures correlated well with those of in-situ hybridization, that is that only the decidua made detectable prolactin, while it was undetectable in the culture medium from trophoblast tissue, irrespective of the stage of pregnancy. This study, for the first time, establishes that only decidualized cells are involved in biosynthesis of prolactin; other prolactin-containing cells in the amnion and trophoblast appear to sequester prolactin, possibly via receptors, suggesting that prolactin may play an important paracrine role within the amnion and syncitio- and cytotrophoblast of the utero-placental unit.

In situ localization of mRNA for epidermal growth factor in the submandibular gland of the mouse.

1985 ◽  
Vol 33 (12) ◽  
pp. 1235-1240 ◽  
Author(s):  
E W Gresik ◽  
R M Gubits ◽  
T Barka
Keyword(s):  
In Situ Hybridization ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Submandibular Gland ◽  
Cdna Probe ◽  
Coding Region ◽  
Specific Hybridization ◽  
Epidermal Growth ◽  
Convoluted Tubules

Epidermal growth factor (EGF) is a polypeptide originally isolated from the mouse submandibular gland, where it is localized immunocytochemically in cells of the granular convoluted tubules (GCT). cDNAs encoding the precursor of mouse submandibular EGF have been cloned (Scott et al. Science 221:236, 1983; Gray et al. Nature 303:722, 1983). A fragment of one of these clones, pmegf10, containing the EGF coding region, was tritium-labeled by nick-translation and used as a probe for in situ hybridization to EGF mRNA. A specific hybridization signal for EGF mRNA was seen only in mature or developing GCT cells. The intensity of the signal was stronger in glands of intact males than in females or in castrated males. In glands of castrates treated with testosterone, or of intact females treated with triiodothyronine (T3), the signal was comparable to that in intact males. In glands of males treated with T3 the intensity of the signal was stronger than in untreated males. A weak to moderate signal was seen in developing GCT cells of 20-day-old males but not females. Hybridization for 3 days gave a stronger signal than that for 1 day. No signal was seen in either sex at 10 days of age, or in control preparations exposed to labeled DNA of pBR322. The presence of EGF mRNA exclusively in GCT cells provides strong evidence that these cells are the only site of synthesis of EGF in the submandibular gland. In situ hybridization with this cDNA probe will provide a sensitive method to determine possible cellular sites of EGF production outside of the submandibular gland.

Increased resolution of in situ hybridization signal by electron microscopy: A comparison with fluorescence microscopy

Genome ◽  
1999 ◽  
Vol 42 (5) ◽  
pp. 1001-1007
Author(s):  
Raouf Fetni ◽  
Patrick Scott ◽  
Frédérique Tihy ◽  
Claude-Lise Richer ◽  
Nicole Lemieux
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
Fluorescence Microscopy ◽  
Dna Sequences ◽  
Cdna Probe ◽  
Single Copy ◽  
Resolving Power ◽  
Chromosomal Structure ◽  
Definition Of

Cytogenetic studies by in situ hybridization (ISH) have proven to be valuable for gene mapping on banded chromosomes when combined with fluorescence microscopy (FISH). However, even under the best conditions, FISH technology has a resolving power inherent to light of just 0.2 µm. Its utilization is further limited by the diffusion of light coming from the fluorescent signal which covers an area considerably larger than the target DNA sequence. The development of new ISH protocols applied to electron microscopy (EMISH) should increase the resolution for cytogenetic mapping and fine chromosomal structure studies. Despite these advances, few attempts have been made which exploit this increased resolution. Here we present a detailed analysis of ISH signals obtained by fluorescence and electron microscopy methodologies to demonstrate and define the higher sensitivity obtainable by electron microscopy. This comparative study was conducted with probes of different origins: telomeric, classical satellite, alpha satellite, and single-copy DNA sequences, which provide a good reference point for later studies. We were also able to map a 200-bp cDNA probe by EMISH. This study assesses the nature of the resolution and the better definition of the EMISH signal, which confirms the greater resolution of electron microscopy as compared with that achieved with light microscopy. It also indicates that better delineation of two closely linked sequences is achieved at the electron microscopy level.Key words: In situ hybridization, electron microscopy, fluorescence microscopy, localization, repetitive and small single-copy probes.

scholarly journals Type IV collagen mRNA accumulates in the mesenchymal compartment at early stages of murine developing intestine.

1990 ◽  
Vol 110 (3) ◽  
pp. 849-857 ◽  
Author(s):  
P Simon-Assmann ◽  
F Bouziges ◽  
J N Freund ◽  
F Perrin-Schmitt ◽  
M Kedinger
Keyword(s):  
In Situ Hybridization ◽  
Type Iv Collagen ◽  
Developmental Stages ◽  
Cdna Probe ◽  
Mesenchymal Cells ◽  
Hybridization Signal ◽  
Collagen Mrna ◽  
Early Stages ◽  
Type Iv

The expression of type IV collagen mRNA during mouse intestinal morphogenesis was examined by in situ hybridization using a cDNA probe corresponding to mRNA for alpha 1 (IV) chain. Type IV collagen mRNA is detected in the embryonic mesenchymal cells at early stages of development (12 d of gestation). A segregation of mesenchymal cells expressing high levels of type IV collagen mRNA in close vicinity of the epithelium occurs just before villus formation. During villus outgrowth, type IV collagen mRNA, still confined to mesenchyme-derived tissues, is progressively restricted to the mucosal connective tissue (the lamina propria) and to a lesser extent to the muscular layers. In the adult, the amount of messenger is quite low as compared to the level found in the developing intestine and the in situ hybridization signal, indistinguishable from the background, is uniform throughout the whole intestinal wall. At all developmental stages no detectable specific hybridization signal is virtually observed over the epithelium cell layer. These results show that high amounts of the type IV collagen messenger are detected during phases of intensive morphogenetic events. Furthermore, they reinforce the notion already gained previously (Simon-Assmann et al. 1988) that the mesenchymal compartment is the principal endogenous source of type IV collagen. They also indicate that the continuous migration of epithelial cells along the basement membrane of intestinal villi in the mature organ is not accompanied by a significant remodeling of the collagen IV network.

scholarly journals Immunohistochemistry and in situ hybridization of P-45017 alpha (17 alpha-hydroxylase/17,20-lyase).

1992 ◽  
Vol 40 (7) ◽  
pp. 903-908 ◽  
Author(s):  
T Suzuki ◽  
H Sasano ◽  
T Sawai ◽  
J I Mason ◽  
H Nagura
Keyword(s):  
In Situ Hybridization ◽  
Leydig Cells ◽  
Cellular Localization ◽  
Cdna Probe ◽  
Simultaneous Detection ◽  
Seminiferous Tubules ◽  
Zona Fasciculata ◽  
Mrna Levels ◽  
Theca Interna

Cytochrome P-45017 alpha catalyzes both 17 alpha-hydroxylation and 17,20-side-chain cleavage in steroidogenesis and lies at a key branch point in the pathways of steroid hormone biosynthesis. To obtain information on the precise localization of P-45017 alpha in swine testis, ovary, and adrenal, we undertook the simultaneous detection of P-45017 alpha mRNA and protein by combining immunohistochemistry with in situ hybridization. In situ hybridization was performed on 4% paraformaldehyde-fixed, paraffin-embedded sections by employing either a 39-base oligomer or a cDNA insert (1.7 KB) of porcine testis P-45017 alpha as DNA probe. Immunohistochemical study was performed by employing anti-P-45017 alpha. Hybridization signals were obtained in Leydig cells of the testis, theca interna of the ovarian follicle, and zona fasciculata reticularis cells of the adrenal cortex. Oligonucleotide probing yielded lower background signal than the cDNA probe. No specific signals were obtained in seminiferous tubules of the testis, medulla, and zona glomerulosa of the adrenal, and in membrana granulosa and interstitial cells of the ovary. Hybridization signals were obtained in the cells where immunoreactivity of the enzyme was observed by immunohistochemistry, except for some Leydig cells of the testis and theca interna cells of the ovary in which only immunoreactivity but not hybridization signal was observed. The present study provided detailed information about the precise cellular localization of P-45017 alpha expression at both the protein and mRNA levels in swine adrenal glands and gonads. This approach of simultaneous immunohistochemistry and in situ hybridization analysis of steroidogenic enzymes can be applied in the future to tissues exhibiting abnormal steroid metabolism and should contribute to a better understanding of steroidogenesis.

scholarly journals Specific detection of neuronal cell bodies: in situ hybridization with a biotin-labeled neurofilament cDNA probe.

1986 ◽  
Vol 34 (7) ◽  
pp. 923-926 ◽  
Author(s):  
P Liesi ◽  
J P Julien ◽  
P Vilja ◽  
F Grosveld ◽  
L Rechardt
Keyword(s):  
In Situ Hybridization ◽  
Cdna Probe ◽  
Neuronal Cell ◽  
Dna Probe ◽  
Specific Detection ◽  
Neurofilament Protein ◽  
Antibody Staining ◽  
Neuronal Cell Bodies ◽  
Specific Mrna

We have used a biotinylated, 300-nucleotide cDNA probe which encodes the 68,000 MW neurofilament protein to detect neurofilament-specific mRNA in situ. The neurofilament message specifically demonstrates the neuronal cell bodies, in contrast to the usual antibody staining which detects their neurites. The hybridization is detected only in neuronal structures. Consequently, detection of the biotinylated neurofilament DNA probe by silver-intensified streptavidin-gold can be specifically used to identify neuronal cell bodies.

scholarly journals Detection of CAIII mRNA in rat skeletal muscle and liver by in situ hybridization.

1991 ◽  
Vol 39 (9) ◽  
pp. 1243-1247 ◽  
Author(s):  
C D Kelly ◽  
N D Carter ◽  
P de Boer ◽  
S Jeffery ◽  
A F Moorman ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
In Situ Hybridization ◽  
Carbonic Anhydrase ◽  
Cdna Probe ◽  
Rat Skeletal Muscle ◽  
Carbonic Anhydrase Iii ◽  
In Situ Methods ◽  
Muscle Section ◽  
Biotinylated Cdna Probe

We carried out a variety of in situ methods of hybridization on rat liver and rat skeletal muscle using 35S-labeled or biotin-labeled rat carbonic anhydrase III (CAIII) cDNA clone. The methods were compared and evaluated. Use of the biotin system produced defined but nonspecific results which were shown not to be due to the biotinylated cDNA probe binding to the mRNA in the muscle sections. This artifact was shown to persist despite various attempts to eliminate it. Alternatively, using 35S-labeled cDNA gave reproducible results which were shown to be consistent with probe binding specifically to mRNA in the muscle section.

scholarly journals In situ localization of amylase mRNA and protein. An investigation of amylase gene activity in normal human parotid gland.

1987 ◽  
Vol 35 (1) ◽  
pp. 9-14 ◽  
Author(s):  
D J Morley ◽  
M E Hodes
Keyword(s):  
In Situ Hybridization ◽  
Cellular Localization ◽  
Cdna Probe ◽  
Immunohistochemical Localization ◽  
Frozen Sections ◽  
Parotid Glands ◽  
Ductal Cells ◽  
Ethanol Acetic Acid ◽  
Mrna And Protein Expression

The distribution of human salivary amylase mRNA was studied by in situ hybridization to a [32P]-labeled amylase cDNA probe. Amylase mRNA was localized to the apical portion of acinar cells in frozen sections of human parotid salivary gland. No hybridization was noted in ductal cells, skeletal muscle, or in connective tissue. These results were consistent with immunohistochemical localization of amylase. The technique of in situ hybridization was modified to permit localization of amylase mRNA in variously fixed, paraffin-embedded parotid glands. Although the hybridization signal decreased with all fixatives, the pattern of localization paralleled that obtained with frozen sections. No advantage was noted in fixation with ethanol-acetic acid or Bouin solution over routine fixation with formalin. These results have important implications for researchers interested in studies of gene expression. We have demonstrated that routinely fixed paraffin blocks of human tissue can be used for cellular localization of specific mRNA. In coordination with immunocytochemistry, in situ hybridization offers a powerful tool for studies of mRNA and protein expression in individual cells.

Presence and distribution of endothelin-1 gene expression in human kidney

1994 ◽  
Vol 267 (4) ◽  
pp. F679-F687 ◽  
Author(s):  
C. Pupilli ◽  
M. Brunori ◽  
N. Misciglia ◽  
C. Selli ◽  
L. Ianni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Renal Cortex ◽  
Cdna Probe ◽  
Renal Medulla ◽  
Human Kidney ◽  
Renal Tumors ◽  
Mrna Levels

To investigate the presence and the distribution of preproendothelin-1 (prepro-ET-1) mRNA in human kidney, eight human kidneys obtained at surgery from patients affected by localized renal tumors were studied. Northern blot analysis using a human prepro-ET-1 cDNA probe labeled with 32P showed the presence of a single band of approximately 2.3 kb that was present both in the renal cortex and medulla of all the kidneys studied. Densitometric analysis of hybridization signals demonstrated that prepro-ET-1 mRNA levels in the renal medulla were 2.2-fold higher than those in the renal cortex. The distribution of prepro-ET-1 mRNA in human kidney was investigated by in situ hybridization using a human prepro-ET-1 RNA probe labeled with 35S. The greatest density of prepro-ET-1 mRNA was observed in the renal medulla, where hybridization signal was demonstrated in vasa recta bundles and capillaries and in collecting ducts. By combining in situ hybridization with immunohistochemical detection of von Willebrand factor, we demonstrated that 93 +/- 2.5% of nontubular medullary cells containing prepro-ET-1 mRNA were endothelial cells. In the cortex, prepro-ET-1 mRNA was localized in the endothelial layer of arcuate and interlobular arteries and veins and in the endothelial cells of afferent arterioles. The results of the present study demonstrate that ET-1 gene expression is present in vascular and tubular structures of the human kidney. It is possible that ET-1 synthesized locally in the human kidney represents a local system affecting renal hemodynamics and functions through paracrine and/or autocrine actions on different renal structures.

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