Localization of alpha-casein gene transcription in sections of epoxy resin-embedded mouse mammary tissues by in situ hybridization.

The objective of our study was to evaluate the suitability of aldehyde-fixed, epoxy resin-embedded tissue for efficient and reproducible detection of casein mRNA in mouse mammary tissue by in situ hybridization. We used mouse alpha-casein-specific, 35S-labeled riboprobes generated from a Gemini-3 vector. Both complementary (anti-sense) and homologous (sense) RNA probes were utilized in our study (specific activity ranged from 5-7 x 10(8) cpm/micrograms). We tested the stability of newly synthesized [3H]-uridine-labeled RNA in tissue sections subjected to epoxy plastic solvents and found that no detectable loss of label occurred during preparation of semi-thin (1-2 micron) plastic sections for situ hybridization. In addition, it was possible to detect alpha-casein mRNA in deplasticized sections of mammary gland tissue taken from normal, pregnant, or lactating mice, pre-neoplastic mammary alveolar hyperplasias, explant cultures, and mammary tumors. A positive hybridization signal was consistently obtained in sections of mammary tissues where the estimated average copy number for total casein mRNA was greater than or equal to 250/cell. In mammary tumors, where the estimated casein mRNA content was much lower (less than 5/cell), our positive hybridization signal occurred in regions of the tumor that, in consecutive sections, stained positive for casein by immunoperoxidase. After formaldehyde-glutaraldehyde fixation, loss of hybridizable RNA from epoxy-embedded tissues and sections appears to be minimal. Image resolution was greatly enhanced over frozen or paraffin sections of mammary tissue. Non-specific binding of the radioactive probes was very low. Protease treatment of the sections was not necessary for detection of hybridizable signal.

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Effect of 3-Methylcholanthrene Administration on Expression of Cytochrome P-450 Isoforms Induced by Phenobarbital in Rat Hepatocytes

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215549804601007 ◽

1998 ◽

Vol 46 (10) ◽

pp. 1151-1160 ◽

Cited By ~ 5

Author(s):

Kazuto Mino ◽

Jun Watanabe ◽

Shinsuke Kanamura

Keyword(s):

In Situ Hybridization ◽

Rat Hepatocytes ◽

Hybridization Signal ◽

Quantitative Immunohistochemistry ◽

Strong Hybridization ◽

Strong Hybridization Signal ◽

Cytochrome P 450

The effects of an inducer on expression of cytochrome P-450 (P-450) isoforms induced antecedently by another inducer are unknown. Thus, we examined the amount of phenobarbital (PB)-inducible P-450 isoforms (P-450 2B1/2B2) in hepatocytes from rats injected first with PB and then with 3-methylcholanthrene (MC) (PB+MC-treated animals) by quantitative immunohistochemistry. In addition, expression of P-450 2B2 mRNA was examined by in situ hybridization. In PB-treated animals, P-450 2B1/2B2 content increased in perivenular and midzonal hepatocytes. In PB+MC-treated animals, however, the PB-induced increase in 2B1/2B2 content was suppressed in perivenular hepatocytes but promoted in midzonal hepatocytes. The hybridization signal for P-450 2B2 mRNA appeared almost exclusively in perivenular hepatocytes after 24 hr of PB injection and disappeared after 48 hr of injection. In PB+MC-treated animals, however, strong hybridization signal was observed in midzonal and perivenular hepatocytes after 48 hr of PB injection. The promotion of the increase in P-450 2B1/2B2 content in midzonal hepatocytes in PB+MC-treated animals probably corresponds to the strong hybridization signal, whereas there appeared to be a divergence between the intensity of the signal and the content in perivenular hepatocytes. The results indicate that MC administration drastically influences the pattern of expression of P-450 isoforms induced by PB in perivenular and midzonal hepatocytes.

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Localization of urokinase-type plasminogen activator messenger RNA in the normal mouse by in situ hybridization.

Journal of Histochemistry & Cytochemistry ◽

10.1177/39.3.1899685 ◽

1991 ◽

Vol 39 (3) ◽

pp. 341-349 ◽

Cited By ~ 64

Author(s):

P Kristensen ◽

J Eriksen ◽

K Danø

Keyword(s):

In Situ Hybridization ◽

Plasminogen Activator ◽

Normal Mouse ◽

Hybridization Signal ◽

Mouse Tissue ◽

Northern Analysis ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Collecting Tubules

The histological distribution of urokinase-type plasminogen activator (u-PA) mRNA was analyzed in normal mouse tissue by in situ hybridization with anti-sense RNA transcribed from three different subclones of a mouse u-PA cDNA. Hybridization signal was found over a distinct fibroblast-like cell in the lamina propria of the gastrointestinal tract, over proximal, distal, and collecting tubules of the kidney, and over the epithelium of the bladder, ductus deferens, and epididymis. No hybridization signal was found over cells of the lung, pancreas, liver, adrenal, pituitary, cerebrum, hypothalamus, cerebellum, sciatic nerve, and striated muscle, nor over endothelial cells in any tissue investigated. The lack of u-PA mRNA in lung tissue was confirmed by Northern analysis and is in contrast to the high amounts of u-PA protein found in this tissue.

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In situ localization of two repetitive DNA sequences to surface-spread pachytene chromosomes of rye

Genome ◽

10.1139/g92-082 ◽

1992 ◽

Vol 35 (4) ◽

pp. 551-559 ◽

Cited By ~ 25

Author(s):

S. M. Albini ◽

T. Schwarzacher

Keyword(s):

In Situ Hybridization ◽

Synaptonemal Complex ◽

Dna Sequences ◽

Meiotic Prophase ◽

Hybridization Signal ◽

Metaphase Chromosomes ◽

Somatic Metaphase ◽

Rdna Signal ◽

Surface Spread

Surface-spread pollen mother cells at meiotic prophase from Secale cereale (rye) were used for fluorescent DNA:DNA in situ localization of two tandemly repeated DNA sequences: pTa71, a wheat rDNA clone, and pSc119.2, a cloned 120-bp repeat from rye heterochromatin. The fluorescent hybridization signal, consisting of many yellow-green dots, was closely associated with the bivalent axes, corresponding to the synaptonemal complex, and located in the surrounding chromatin. The rDNA signal was associated with one bivalent, the smallest of the seven, at a distance about 13% of the bivalent length from the telomere. This corresponded to the position of the nucleolar organizing region of silver-stained synaptonemal complexes analyzed under the electron microscope and published data for somatic metaphase chromosomes. The relative length of the axis covered with the rDNA signal is less than expected from somatic metaphases, but it corresponds more closely to the proportion of the sequences in the genome. The hybridization signal with the 120-bp repeat was located mainly at the telomeric regions of several bivalents that showed thickenings of the axis after DAPI staining, probably corresponding to somatic C-bands. These major and some minor intercalary sites agree with the distribution of the 120-bp repeat in somatic metaphase. Fluorescent in situ hybridization to plant surface-spread pachytene chromosomes, which can be obtained in large numbers, has great potential for studying meiotic prophase, high-resolution mapping of DNA sequences, and investigating the relationship of DNA sequences to the synaptonemal complex.Key words: in situ hybridization, cereals, pachytene, meiosis, synaptonemal complex, physical mapping.

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

The Journal of Cell Biology ◽

10.1083/jcb.110.3.849 ◽

1990 ◽

Vol 110 (3) ◽

pp. 849-857 ◽

Cited By ~ 57

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.

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Sex chromosome composition revealed in Characidium fishes (Characiformes: Crenuchidae) by molecular cytogenetic methods

Biologia ◽

10.2478/s11756-014-0434-0 ◽

2014 ◽

Vol 69 (10) ◽

Cited By ~ 2

Author(s):

Marlon Pazian ◽

Claudio Oliveira ◽

Fausto Foresti

Keyword(s):

In Situ Hybridization ◽

Sequence Analysis ◽

Sex Chromosome ◽

Repetitive Sequences ◽

Pericentromeric Region ◽

W Chromosome ◽

Degenerate Oligonucleotide ◽

Clone Sequence ◽

Positive Hybridization

AbstractThe W chromosome of the fishes Characidium cf. fasciatum, Characidium sp. and Characidium cf. gomesi is heterochromatic, as is usually seen in most Characidium species. Samples of W-chromatin were collected by mechanical microdissection and amplified by DOP-PCR (degenerate oligonucleotide-primed polymerase chain reaction), to be used as painting probes (DCg and CgW) and for sequence analysis. FISH (fluorescence in situ hybridization) with DCg probe painted the whole W chromosome, the pericentromeric region of Z chromosomes and the terminal region of B chromosomes. DOP-PCR-generated fragments were cloned, sequenced and tested by in situ hybridization, but only CgW4 produced positive hybridization signals. Clone sequence analysis recovered seven distinct sequences, of which six did not reveal any similarity to other known sequences in the GenBank or GIRI databases. Only CgW9 clone sequence was recognized as probably derived from a Helitron-transposon similar to that found in the genome of the zebrafish Danio rerio. Our results show that the composition of Characidium’s W chromosome does seem rich in repetitive sequences as well as other W chromosomes found in several species with a ZW sex-determining mechanism.

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In Situ Hybridization Signal Patterns in Recurrent Laryngeal Squamous Papillomas Indicate that HPV Integration Occurs at an Early Stage

Head and Neck Pathology ◽

10.1007/s12105-011-0308-5 ◽

2011 ◽

Vol 6 (1) ◽

pp. 32-37 ◽

Cited By ~ 4

Author(s):

Erin Grace Brooks ◽

Mark Francis Evans ◽

Christine Stewart-Crawford Adamson ◽

Zhihua Peng ◽

Vanitha Rajendran ◽

...

Keyword(s):

In Situ Hybridization ◽

Early Stage ◽

Hybridization Signal

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The random primer labeling technique applied to in situ hybridization on tissue sections.

Journal of Histochemistry & Cytochemistry ◽

10.1177/36.10.3138309 ◽

1988 ◽

Vol 36 (10) ◽

pp. 1335-1340 ◽

Cited By ~ 13

Author(s):

M Thomas-Cavallin ◽

O Aït-Ahmed

Keyword(s):

Drosophila Melanogaster ◽

In Situ Hybridization ◽

Specific Activity ◽

High Specific Activity ◽

High Sensitivity ◽

Random Primer ◽

Tissue Sections ◽

In Situ Hybridizations ◽

Labeling Method

We report an application of the random primer labeling technique to in situ hybridizations on tissue sections. The ease of the method and the high specific activity achieved make it valuable when a large number of probes must be analyzed and high sensitivity is needed. We have applied this technique to study the spatial expression of a cluster of maternally acting genes (the yema gene region of Drosophila melanogaster which encodes eleven transcripts, some of them having a very low level of expression) (Aït-Ahmed et al., 1978: Dev Biol 122:153; Aït-Ahmed et al., unpublished results). The results reported here concern one of the transcripts of the yema region, which displays a peculiar anterior localization in the oocyte. We demonstrate that the "oligo-labeling" method allows a far better level of detection of the transcript of interest.

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