In situ transcription: specific synthesis of complementary DNA in fixed tissue sections

Science ◽  
1988 ◽  
Vol 240 (4859) ◽  
pp. 1661-1664 ◽  
Author(s):  
L. Tecott ◽  
J. Barchas ◽  
J. Eberwine
Keyword(s):  
Fixed Tissue ◽  
Complementary Dna ◽  
Tissue Sections ◽  
Specific Synthesis

Enhanced DNA fragmentation in the thymus of spontaneously hypertensive rats

1999 ◽  
Vol 276 (6) ◽  
pp. H2135-H2140 ◽  
Author(s):  
Hidekazu Suzuki ◽  
Frank A. Delano ◽  
Neema Jamshidi ◽  
Dan Katz ◽  
Mikiji Mori ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Spontaneously Hypertensive Rat ◽  
Organ Injury ◽  
Fixed Tissue ◽  
Thymic Atrophy ◽  
Spontaneously Hypertensive ◽  
Tissue Sections ◽  
Dna Nicking ◽  
Wistar Kyoto

The mechanisms contributing to organ injury in hypertension have been incompletely defined. The thymus gland of the spontaneously hypertensive rat (SHR) shows significant atrophy at the age of 15 wk compared with its normotensive control, the Wistar-Kyoto rat (WKY). The aim of the present study was to examine the thymus of SHR for evidence of DNA nicking as one of the mechanisms for thymic atrophy. SHR and WKY were subjected to adrenalectomy or sham surgery at 12 wk and studied at 15 wk. Adrenalectomy served to normalize the blood pressure in the SHR. DNA nicking was detected by in situ nick-end labeling (ISEL) of fixed tissue sections. Tissue sections were treated with proteolysis, and terminal deoxyribonucleotidyl transferase was used to incorporate biotinylated deoxynucleotides into DNA nick end in situ. Separately, DNA fragmentation was evaluated by measuring the level of released mono- and oligonucleosomes to the cytoplasm. A higher number of thymic ISEL-positive cells and a higher level of cytoplasmic mono- and oligonucleosomes were observed in SHR than in WKY. After adrenalectomy the enhanced level of ISEL and cytoplasmic mono- and oligonucleosomes in SHR was reduced to the level in WKY. Dexamethasone treatment (0.05 mg ⋅ kg−1⋅ day−1) in WKY serves to decrease the thymus weight and significantly elevate the level of mono- and oligonucleosomes. Thus increased DNA fragmentation represents one of the mechanisms associated with thymic atrophy, a feature that reflects immune suppression in SHR.

scholarly journals Combined in Situ Zymography, Immunofluorescence, and Staining of Iron Oxide Particles in Paraffin-Embedded, Zinc-Fixed Tissue Sections

2012 ◽  
Vol 11 (5) ◽  
pp. 7290.2011.00055 ◽  
Author(s):  
Eyk Schellenberger ◽  
Akvile Haeckel ◽  
Lena Schoenzart ◽  
Franziska Appler ◽  
Joerg Schnorr ◽  
...  
Keyword(s):  
Iron Oxide ◽  
Iron Oxide Particles ◽  
Fixed Tissue ◽  
Tissue Sections ◽  
Oxide Particles ◽  
In Situ Zymography

scholarly journals A simple, reliable, and sensitive method for nonradioactive in situ hybridization: use of microwave heating to improve hybridization efficiency and preserve tissue morphology.

1996 ◽  
Vol 44 (3) ◽  
pp. 281-287 ◽  
Author(s):  
H Y Lan ◽  
W Mu ◽  
Y Y NG ◽  
D J Nikolic-Paterson ◽  
R C Atkins
Keyword(s):  
In Situ Hybridization ◽  
Microwave Oven ◽  
Hybridization Signal ◽  
General Applicability ◽  
Fixed Tissue ◽  
Microwave Pretreatment ◽  
Tissue Sections ◽  
Hybridization Time ◽  
Protease Treatment

The digestion of fixed tissue sections is a critical step in the optimization of any in situ hybridization protocol. We describe a novel application of microwave oven heating to optimize mRNA detection in paraformaldehyde-fixed tissues by in situ hybridization using digoxigenin-labeled probes. This technique replaces protease digestion of fixed tissue sections with 10 min of microwave pretreatment, followed by either conventional hybridization or hybridization involving microwave incubation. This new technique has several advantages over the standard protease treatment-based methods presently in use. (a) Microwave oven heating is a simple, rapid, and highly reproducible technique. (b) Microwave pretreatment significantly increased the hybridization signal and reduced the background compared to conventional protease digestion. Consequently, the hybridization time required to obtain optimal mRNA detection was reduced to 30 min. (c) Ten minutes of microwave pretreatment produced an optimal hybridization signal in six different tissues using a variety of probes, demonstrating the general applicability of this technique. (d) Microwave heating of the probe during the hybridization step itself further reduced the hybridization time and substantially enhanced the hybridization signal obtained from proteinase K-digested tissue. (e) Microwave pretreatment caused no discernible loss of fine cell structure and tissue morphology compared to untreated tissue sections. In conclusion, microwave oven heating can replace the complicated strategies and poor reproducibility of protease treatment of tissue sections, resulting in a simple, rapid, more reliable and sensitive method that has general applicability for in situ hybridization.

HCR of raphe serotonergic neurons in fixed tissue sections  v1

2021 ◽  
Author(s):  
Alex Buckley
Keyword(s):  
In Situ Hybridization ◽  
Brain Tissue ◽  
Mouse Brain ◽  
Fluorescent In Situ Hybridization ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Serotonergic Neurons ◽  
Fixed Tissue ◽  
Tissue Sections

This is an RNA fluorescent in-situ hybridization (FISH) protocol that utilizes hybridization chain reaction technology from Molecular Instruments. The protocol fluorescently labels different mRNAs (up to 4 different mRNAs) such that they become suitable for imaging. This protocol is designed specifically for fixed mouse brain tissue sections that contain raphe serotonergic neurons, but can be applied to other regions of the mouse brain as well.

scholarly journals Preservation of RNA for in situ hybridization: Carnoy's versus formaldehyde fixation.

1992 ◽  
Vol 40 (12) ◽  
pp. 1879-1885 ◽  
Author(s):  
S Urieli-Shoval ◽  
R L Meek ◽  
R H Hanson ◽  
M Ferguson ◽  
D Gordon ◽  
...  
Keyword(s):  
Molecular Weight ◽  
In Situ Hybridization ◽  
Organic Solvent ◽  
High Molecular Weight ◽  
Fixed Tissue ◽  
Rna Integrity ◽  
Tissue Sections ◽  
Carnoy’S Solution ◽  
Rna Content

Tissues fixed with organic solvent fixatives such as Carnoy's solution are known to give poor and erratic results with in situ hybridization, whereas those fixed with paraformaldehyde produce more consistent results. To understand this difference and to improve the utility of Carnoy's-fixed tissue for in situ hybridization, we explored several parameters of RNA integrity and preservation. Carnoy's-fixed, paraffin-embedded livers and paraformaldehyde-fixed, paraffin-embedded livers of mice were compared for RNA extractability, degradation, and hybridizability. In addition, retention of RNA in tissue sections after sequential in situ hybridization treatments was compared. RNA was found to be easily extractable from Carnoy's-fixed liver and was well preserved, with only slight degradation of high molecular weight RNA. Conversely, only a small percentage of the RNA was extractable from paraformaldehyde-fixed liver unless the tissue was digested with protease. The extracted RNA was well preserved, without detectable degradation. Sections of tissue fixed in Carnoy's solution subjected to in situ hybridization retained only about 10% of their original RNA content and gave correspondingly weak in situ hybridization signals. Formaldehyde-fixed tissues retained much more of the RNA (about 45%) and produced strong in situ hybridization signals. Treatment of Carnoy's-fixed tissue sections with vaporous formaldehyde increased retention of RNA and provided in situ hybridization signals comparable with those of paraformaldehyde-fixed tissues.

Flat embeddment of monolayer cells, tissue sections, and other cellular materials attached onto glass substrate

1990 ◽  
Vol 48 (3) ◽  
pp. 766-767
Author(s):  
Jeffrey P. Chang ◽  
Jaang J. Wang
Keyword(s):  
Present Report ◽  
Cellular Materials ◽  
Cover Glass ◽  
Tissue Sections ◽  
Embedding Technique ◽  
Exfoliated Cells ◽  
Black Paper ◽  
Cell Concentrations ◽  
Flat Embedding

Flat embeddment of certain specimens for electron microscopy is necessary for three classes of biological materials: namely monolayer cells, tissue sections of paraffin or plastics, as well as cell concentrations, exfoliated cells, and cell smears. The present report concerns a flat-embedding technique which can be applied to all these three classes of materials and which is a modified and improved version of Chang's original methodology.Preparation of coverglasses and microslides. Chemically cleaned coverglasses, 11 × 22 mm or other sizes, are laid in rows on black paper. Ink-mark one coner for identifying the spray-side of the glass for growing cells. Lightly spray with Teflon monomer (Heddy/Contact Inductries, Paterson, NO 07524, U.S.A.) from a pressurized can. Bake the sprayed glasses at 500°F for 45 min on Cover-Glass Ceramic Racks (A. Thomas Co. Philadelphia), for Teflon to polymerize.Monolayer Cells. After sterilization, the Teflon-treated coverglasses, with cells attached, are treated or fixed in situ in Columbia staining dishes (A. Thomas Co., Philadelphia) for subsequent processing.

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