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.

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 Achieving Good Protection on Ultra-High Molecular Weight Polythene by In Situ Growth of Amorphous Carbon Film

Coatings ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 584
Author(s):  
Rui Dang ◽  
Liqiu Ma ◽  
Shengguo Zhou ◽  
Deng Pan ◽  
Bin Xia
Keyword(s):  
Molecular Weight ◽  
Epitaxial Growth ◽  
High Molecular Weight ◽  
Amorphous Carbon ◽  
Transition Layer ◽  
In Situ Growth ◽  
Good Protection ◽  
Ultra High Molecular Weight ◽  
Carbon Plasma

Ultra-high molecular weight polythene (UHMWPE), with outstanding characteristics, is widely applied in modern industry, while it is also severely limited by its inherent shortcomings, which include low hardness, poor wear resistance, and easy wear. Implementation of feasible protection on ultra-high molecular weight polythene to overcome its shortcomings would be of significance. In the present study, amorphous carbon (a-C) film was fabricated on ultra-high molecular weight polythene (UHMWPE) to provide good protection, and the relevant growth mechanism of a-C film was revealed by controlling carbon plasma currents. The results showed the in situ transition layer, in the form of chemical bonds, was formed between the UHMWPE substrate and the a-C film with the introduction of carbon plasma, which provided strong adhesion, and then the a-C film continued epitaxial growth on the in situ transition layer with the treatment of carbon plasma. This in situ growth of a-C film, including the in situ transition layer and the epitaxial growth layer, significantly improved the wetting properties, mechanical properties, and tribological properties of UHMWPE. In particular, good protection by in situ growth a-C film on UHMWPE was achieved during sliding wear.

scholarly journals Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. II. Lectin binding to the isolated glycoproteins of normal and malignant gastric mucosa.

1984 ◽  
Vol 32 (7) ◽  
pp. 690-696 ◽  
Author(s):  
J Fischer ◽  
G Uhlenbruck ◽  
P J Klein ◽  
M Vierbuchen ◽  
R Fischer
Keyword(s):  
Molecular Weight ◽  
Gastric Mucosa ◽  
High Molecular Weight ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Gastric Cancer Cells ◽  
Tissue Sections ◽  
Gradient Gel Electrophoresis ◽  
Triton X

Using affinity chromatography on HPA-, PNA-, Con A, and WGA-agarose columns only a part (10-30%) of the high molecular weight mucous glycoproteins could be isolated from the Triton X-100 solubilized components of normal as well as carcinomatous gastric mucosa. The main part of the mucus was not bound by the lectins, which corresponds to our earlier lectin histochemical observations on paraffin-embedded tissue sections. The lectin-bound mucous glycoproteins had a relatively lower molecular weight, ranging from about 250-1,000 kilodaltons, as indicated by polyacrylamide gradient gel electrophoresis and by gel filtration on Biogel A 1.5 m column. In gas chromatographic analysis the molar ratio of aminohexoses to galactose was found to be much higher (3:1) in the lectin-bound mucous substances than in the whole high molecular weight mucus (1:1). This finding indicates that lectins have a higher affinity to the hexosamine rich components of mucus, which may be special forms of mucous glycoprotein molecules or the incompletely glycosylated core and backbone regions of the oligosaccharide chains of mucus. Extremely high hexosamine values (10:1) were found in the PNA isolated mucus of gastric adenocarcinoma. Since it is known that PNA binds to the terminal disaccharide, beta-galactose-(1-3)-N-acetylgalactosamine, which is localized at the reducing end of the oligosaccharide chains of mucus, it is highly probable that the elongation of the oligosaccharide side chains is disturbed in gastric cancer cells.

Wear behavior of in situ polymerized carbon nanotube/ultra high molecular weight polyethylene composites

2013 ◽  
Vol 21 (9) ◽  
pp. 965-970 ◽  
Author(s):  
Hong-Jo Park ◽  
Jihun Kim ◽  
Yongsok Seo ◽  
Junho Shim ◽  
Moon-Yong Sung ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Carbon Nanotube ◽  
High Molecular Weight ◽  
Wear Behavior ◽  
Ultra High Molecular Weight ◽  
Polyethylene Composites
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