Six μm-thick paraffin sections were mounted on Super frost + glass slides and deparaffinized. The slides were mounted onto flow through slide chambers and placed in a Tecan Freedom Evo automated hybridization instrument (Tecan, Männedorf, Switzerland) in which the following steps were performed: proteinase-K treatment 15 μg/ml at 37°C for 8 min, pre-hybridization in Exiqon hybridization buffer (Exiqon, Vedbæk, Denmark) at 62°C for 15 min, hybridization with 40 nM miR-21 probe, stringent washes with 5 × SSC, 1 × SSC and 0.2 × SSC buffers at 62°C over 33 min, DIG blocking reagent (Roche, Mannheim, Germany) in maleic acid buffer containing 2% sheep serum at 30°C for 15 min, alkaline phosphatase-conjugated anti-digoxigenin (diluted 1:500 in blocking reagent, Roche) at 30°C for 30 min, enzymatic development using 4-nitro-blue tetrazolium (NBT) and 5-brom-4-chloro-3′-Indolylphosphate (BCIP) substrate (Roche) forming dark-blue NBT-formazan precipitate at 30°C for 60 min, nuclear fast red counterstain (Vector Laboratories, Burlingname, CA), at 25°C for 1 min. The slides were then dismantled in water, dehydrated in alcohol solutions and mounted with eukitt mounting medium (VWR, Herlev, Denmark). For each patient, two slides were hybridized with the full length miR-21 probe. To minimize day–day variations all probes were pre-diluted in hybridization buffer in quantities determined for a single experiment. The same proteinase-K stock was used through-out the experimental period. The following steps were standardized in all steps on different experimental days: tissue sectioning at 6 μm, incubation times, incubation temperatures, pre-diluted probes, antibody dilutions, proteinase-K batch. For probe specificity analysis the full length miR-21 probe was replaced with the 5′-end probe, 3′-end probe, 3-mismatch probe, scrambled probe (all at 40 nM), or the probe for U6 snRNA (0.1 nM). For fluorescent ISH, the sections were processed as above, except that detection of the DIG-labeled probes was done with peroxidase-conjugated sheep anti-DIG (Roche) followed by TSA-FITC substrate (Invitrogen, Taastrup, Denmark) according to manufacturer’s recommendations. Slides were coverslipped using DAPI mounting media (Invitrogen).
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Maleic acid
Maleic acid
Maleic acid is a dicarboxylic acid with the chemical formula C4H4O4.
It is a white, crystalline solid commonly used in the production of various chemicals and materials.
Maleic acid has a wide range of applications, including the synthesis of resins, plasticizers, and pharmaceuticals.
It is also used in the production of maleic anhydride, which is a precursor to many important industrial chemicals.
Maleic acid is an important intermediate in the chemical industry and has been the focus of extensive research and development efforts.
Desription may contain 1 typo for authenticity..
It is a white, crystalline solid commonly used in the production of various chemicals and materials.
Maleic acid has a wide range of applications, including the synthesis of resins, plasticizers, and pharmaceuticals.
It is also used in the production of maleic anhydride, which is a precursor to many important industrial chemicals.
Maleic acid is an important intermediate in the chemical industry and has been the focus of extensive research and development efforts.
Desription may contain 1 typo for authenticity..
Most cited protocols related to «Maleic acid»
250 μl of thawed hydrolysate and 15 μl of sorbitol (0.1000 g/100 ml aqueous) were transferred to a vial and concentrated to dryness under a stream of N2. The internal standard was added to correct for subsequent differences in derivatization efficiency and changes in sample volume during heating. Dried extracts were dissolved in 500 μl of silylation–grade acetonitrile followed by the addition of 500 μl N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) (Thermo Scientific, Bellefonte, PA), and samples then heated for 1 h at 70°C to generate trimethylsilyl (TMS) derivatives [43 (link)]. After 1 day, 1-μl aliquots were injected into an Agilent Technologies Inc. (Santa Clara, CA) 5975C inert XL gas chromatograph-mass spectrometer, fitted with an Rtx-5MS with Integra-guard (5% diphenyl/95% dimethyl polysiloxane) 30 m × 250 μm × 0.25 μm film thickness capillary column. The standard quadrupole GCMS was operated in the electron ionization (EI) (70 eV) mode, with 6 full-spectrum (50–650 Da) scans per second. Gas (helium) flow was 1.33 ml per minute with the injection port configured in the splitless mode. The injection port, MS Source, and MS Quad temperatures were 250°C, 230°C, and 150°C, respectively. The initial oven temperature was held at 50°C for 2 min and was programmed to increase at 20°C per min to 325°C and held for another 11 min, before cycling back to the initial conditions. A large user-created database (>1600 spectra) of mass spectral EI fragmentation patterns of TMS-derivatized compounds, as well as the Wiley Registry 8th Edition combined with NIST 05 mass spectral database, were used to identify the metabolites of interest to be quantified. Peaks were reintegrated and reanalyzed using a key selected ion, characteristic m/z fragment, rather than the total ion chromatogram, to minimize integrating co-eluting metabolites. The extracted peaks of known metabolites were scaled back up to the total ion current using predetermined scaling factors. Unidentified metabolites used the scaling factor for the internal standard (sorbitol) and were denoted by their RT as well as key m/z fragments. The mass-to-charge ratios used as extracted ions were as follows: iso-sinapyl alcohol (354), iso-sinapic acid (368), iso-syringin (354), 5-hydroxyconiferyl alcohol-4-O-glucoside (412), 5-hydroxyconiferyl alcohol-4-O-glucoside (412), 3,4-dihydroxybenzoic acid (370), xanthine (368), hypoxanthine (265), succinic acid (247), guanosine (324), uracil (241), citraconic acid (259), guanine (352), 5-hydroxyferulic acid (411), uridine (258), maleic acid (245), secoisolariciresinol (560), 5-oxo-proline (156), adenine (264), 1-O-trans-feruloylglycerol (249), vanillin (297, 194), ferulic acid (338), adenosine (236), p-coumaric acid (308), caffeic acid (396), p-hydroxybenzaldehyde (392, 194), coniferyl alcohol (324), 5-hydroxyconiferyl alcohol (412), coniferyl aldehyde (323), guaiacylglycerol (297), sinapyl aldehyde (353), syringylglycerol (327), p-hydroxyphenylpyruvic acid (396), syringaresinol (327), pinoresinol (502), hydroxymethylfurfural (183). Peaks were quantified by area integration and the concentrations were normalized to the quantity of the internal standard recovered, volume of sample extracted, derivatized, and injected.
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Isolated DNA (3 μg of each sample) was digested with different combinations of REs (as described in the figures) and then separated on a 0.7% agarose gel at 2 V/cm for 16 h. The telomere signals were detected by Southern blot analysis as previously indicated37 (link). In brief, after DNA was transferred from gel to a positive-charged nylon membrane, the transferred DNA was fixed by UV crosslinking. The cross-linked membrane was then hybridized with the hypersensitive DIG-labeled telomere probe overnight at 42 °C. After hybridization, the membrane was washed with buffer 1 (2× SSC, 0.1% SDS) at room temperature for 15 min and then washed twice with buffer 2 (0.5× SSC, 0.1% SDS) at 55 °C for 15 min. Next, the membrane was washed with DIG wash buffer (1× maleic acid buffer with 0.3% Tween-20) for 5 min. Then the membrane was incubated with 1× DIG blocking solution for 30 min at room temperature. After blocking, the membrane was incubated with anti-DIG antibody (Roche) in 1× blocking solution (1 to 10,000 dilution) for 30 min at room temperature. The membrane was then washed with DIG buffer two times at room temperature for 15 min. After washing, telomere signals were detected by incubating with CDP-star for 5 min. Image quantification was performed using Image Quant software to measure the intensity value of each telomere smear. The intensity value of each sample was then adjusted with the background graphed from a lane with no DNA sample66 .
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Antibodies, Anti-Idiotypic
Buffers
Crossbreeding
Hypersensitivity
maleic acid
Nylons
Sepharose
Southern Blotting
Technique, Dilution
Telomere
Tissue, Membrane
Tween 20
Placentae were dissected in cold phosphate buffered saline (PBS) and fixed overnight in 4% paraformaldehyde in PBS at 4°C. After rinsing in PBS, tissues were processed through sucrose gradients (10% in PBS and 25% in PBS) before being embedded in OCT (Tissue Tek). Ten μm sections were cut and mounted on Super Frost Plus slides (VWR) and stored at -80°C. For in situ hybridization, sections were re-hydrated in PBS, post-fixed in 4% paraformaldehyde for 10 minutes, treated with proteinase K (30 μg/ml) for 10 minutes at room temperature, acetylated for 10 minutes (acetic anhydride, 0.25%; Sigma) and hybridized with digoxigenin-labeled probes overnight at 65°C. Digoxigenin labeling was done according to the manufacturers instructions (Roche). Hybridization buffer contained 1× salts (200 mM sodium choride, 13 mM tris, 5 mM sodium phosphate monobasic, 5 mM sodium phosphate dibasic, 5 mM EDTA), 50% formamide, 10% dextran sulfate, 1 mg/ml yeast tRNA (Roche), 1× Denhardt's (1% w/v bovine serum albumin, 1% w/v Ficoll, 1% w/v polyvinylpyrrolidone), and DIG-labeled probe (final dilution of 1:2000 from reaction with 1 μg template DNA). Two 65°C post-hybridization washes (1× SSC, 50% formamide, 0.1% tween-20) followed by two room temperature washes in 1× MABT (150 mM sodium chloride, 100 mM maleic acid, 0.1% tween-20, pH7.5) were followed by 30 minutes RNAse treatment (400 mM sodium chloride, 10 mM tris pH7.5, 5 mM EDTA, 20 μg/ml RNAse A). Sections were blocked in 1× MABT, 2% blocking reagent (Roche), 20% heat inactivated goat serum for 1 hour and incubated overnight in block with anti-DIG antibody (Roche) at a 1:2500 dilution at 4°C. After four 20 minute washes in 1× MABT, slides were rinsed in 1× NTMT (100 mM NaCl, 50 mM MgCl, 100 mM tris pH 9.5, 0.1% tween-20) and incubated in NBT/BCIP in NTMT according to the manufacturers instructions (Promega). Slides were counterstained with nuclear fast red, dehydrated and cleared in xylene and mounted in cytoseal mounting medium (VWR).
For double labeled in situ hybridizations, a fluorescein-labeled probe was generated following the manufacturers instructions (Roche) and added to the hybridization mix along with the DIG-labeled probe. Following NBT/BCIP development of the first probe, the antibody-enzyme conjugate was inactivated by 30 min incubation in 1× MABT at 65°C, followed by 30 min in 0.1 M glycine pH 2.2 at room temperature. The slides were then blocked again in 1× MABT, 2% blocking reagent (Roche), 20% heat inactivated goat serum for 1 hour and incubated overnight in block with anti-Fluorescein antibody (Roche) at a 1:2500 dilution at 4°C. The second signal was developed as previously described but one modification, the substrate INT/BCIP (Roche), producing a brown colour, was used in place of NBT/BCIP. As the INT/BCIP is soluble in alcohols and xylene, slides were counterstained with nuclear fast red and mounted directly under Crystal Mount Aqueous Mounting Medium (Sigma).
For double labeled in situ hybridizations, a fluorescein-labeled probe was generated following the manufacturers instructions (Roche) and added to the hybridization mix along with the DIG-labeled probe. Following NBT/BCIP development of the first probe, the antibody-enzyme conjugate was inactivated by 30 min incubation in 1× MABT at 65°C, followed by 30 min in 0.1 M glycine pH 2.2 at room temperature. The slides were then blocked again in 1× MABT, 2% blocking reagent (Roche), 20% heat inactivated goat serum for 1 hour and incubated overnight in block with anti-Fluorescein antibody (Roche) at a 1:2500 dilution at 4°C. The second signal was developed as previously described but one modification, the substrate INT/BCIP (Roche), producing a brown colour, was used in place of NBT/BCIP. As the INT/BCIP is soluble in alcohols and xylene, slides were counterstained with nuclear fast red and mounted directly under Crystal Mount Aqueous Mounting Medium (Sigma).
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Acid Hybridizations, Nucleic
Antibodies, Anti-Idiotypic
Cardiac Arrest
Diethyl Pyrocarbonate
Digoxigenin
Domestic Sheep
Edetic Acid
Embryo
Endopeptidase K
Fluorescein
formamide
Magnesium Chloride
maleic acid
Methanol
Phosphates
RNA polymerase SP6
RNA Probes
Saline Solution
Serum
Sodium Chloride
Sodium Citrate
Squalidae
Sulfate, Dextran
Technique, Dilution
Transcription, Genetic
Transfer RNA
Tromethamine
Tween 20
Yeasts
Most recents protocols related to «Maleic acid»
The Coconut shells were obtained from Mombasa County -Kenya and transported to the Kenyatta University laboratory. Glycerol, potassium manganate VII, Sodium hydroxide, and sulphuric acid were obtained from Kenya Science Chemical Limited (Kenya), while maleic acid was purchased from Sigma Aldrich Company (Germany).
The mass balance of PUF acidolysis was
obtained by comparing the total weight of gas, liquid, and solid products
versus the starting weight of PUF and maleic acid. The overall yield
of repolyol from PUF acidolysis after hydrolysis and purification
was calculated based on the obtained neat repolyol versus the polyol
content within the input PUF.
obtained by comparing the total weight of gas, liquid, and solid products
versus the starting weight of PUF and maleic acid. The overall yield
of repolyol from PUF acidolysis after hydrolysis and purification
was calculated based on the obtained neat repolyol versus the polyol
content within the input PUF.
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Conversion of anhydride to acid was performed by hydrolysis in NaOH. Here 800 mg of polymer was dissolved in 2 g of KOH, 20 mL of water in a 100 mL round botom and reflux the mixture for 4 h. This initially dissolved the polymer and at the end, reaction mixture becomes clear. The hydrolyzed polymer was subjected to dialysis using 3.5 KDa cut off membrane for 36 h. After that the polymer was precipitated by the addition of water and HCl(aq). Centrifuging the precipitate was followed by decanting off the supernatant. Wash the precipitate using 0.1 N HCl water three times and dry the precipitate using nitrogen. A brownish solid polymer material was collected.
The acidolysis reaction
was carried out in a glass reaction system. In this setup, a 250 mL
round-bottom flask was connected to a coldfinger that was connected
through Tygon tubing to either a gas evolution buret or a filtration
flask with saturated calcium hydroxide (Ca(OH)2) solution.
The liquid products were collected in a round-bottom flask while evolved
carbon dioxide (CO2) gas captured by the gas evolution
buret or isolated as calcium carbonate (CaCO3) in the filtration
flask. Saturated Ca(OH)2 was prepared by mixing excess
CaO with DI water at room temperature. For each reaction, ground PUF
was premixed with maleic acid in the round-bottom flask, and the reaction
mixture was purged with N2. Magnetic stirring at 250 rpm
and heat through a regulated oil bath were applied. After the reaction,
the product mixture was cooled to room temperature, washed with EtOAc,
and vacuum-filtered through a Buchner funnel equipped with filter
paper. The solid residue (if any) was collected and dried under vacuum.
The liquid filtrate was transferred to a 50 mL centrifuge tube for
product separation.
was carried out in a glass reaction system. In this setup, a 250 mL
round-bottom flask was connected to a coldfinger that was connected
through Tygon tubing to either a gas evolution buret or a filtration
flask with saturated calcium hydroxide (Ca(OH)2) solution.
The liquid products were collected in a round-bottom flask while evolved
carbon dioxide (CO2) gas captured by the gas evolution
buret or isolated as calcium carbonate (CaCO3) in the filtration
flask. Saturated Ca(OH)2 was prepared by mixing excess
CaO with DI water at room temperature. For each reaction, ground PUF
was premixed with maleic acid in the round-bottom flask, and the reaction
mixture was purged with N2. Magnetic stirring at 250 rpm
and heat through a regulated oil bath were applied. After the reaction,
the product mixture was cooled to room temperature, washed with EtOAc,
and vacuum-filtered through a Buchner funnel equipped with filter
paper. The solid residue (if any) was collected and dried under vacuum.
The liquid filtrate was transferred to a 50 mL centrifuge tube for
product separation.
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In each 50 mL
centrifuge tube, the
liquid products were dissolved in 30 mL of EtOAc solution and mixed
with 20 mL of NaOH aqueous solution (50 wt %/wt, aq). Prior to centrifugation,
the two liquid phases were well mixed by a mechanical shaker for 5
min to enhance product extraction. The centrifugation was performed
at 7000 rpm for 10 min. Next, the liquid in the tube separated into
three phases; the top EtOAc phase contained amides and some repolyol
products, the middle phase was repolyol with some water-EtOAc mixture,
and the bottom aqueous phase contained leftover excess maleic acid,
amides, and other byproducts. The top phase could be further purified
by repeating the centrifugation process with a NaOH solution to remove
amides from the EtOAc phase. After purification, the repolyol from
the top EtOAc phase and the middle phase were combined. The repolyol
product was dried by rotary evaporation to remove residual EtOAc and
water. The byproducts in the aqueous phase were also collected by
removing water through a rotovap. The repolyol and product in the
aqueous phase were analyzed.
centrifuge tube, the
liquid products were dissolved in 30 mL of EtOAc solution and mixed
with 20 mL of NaOH aqueous solution (50 wt %/wt, aq). Prior to centrifugation,
the two liquid phases were well mixed by a mechanical shaker for 5
min to enhance product extraction. The centrifugation was performed
at 7000 rpm for 10 min. Next, the liquid in the tube separated into
three phases; the top EtOAc phase contained amides and some repolyol
products, the middle phase was repolyol with some water-EtOAc mixture,
and the bottom aqueous phase contained leftover excess maleic acid,
amides, and other byproducts. The top phase could be further purified
by repeating the centrifugation process with a NaOH solution to remove
amides from the EtOAc phase. After purification, the repolyol from
the top EtOAc phase and the middle phase were combined. The repolyol
product was dried by rotary evaporation to remove residual EtOAc and
water. The byproducts in the aqueous phase were also collected by
removing water through a rotovap. The repolyol and product in the
aqueous phase were analyzed.
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Top products related to «Maleic acid»
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Maleic acid is a dicarboxylic acid chemical compound used in various laboratory and industrial applications. It serves as a precursor for the synthesis of other chemical compounds and as a reagent in analytical procedures. Maleic acid has a molecular formula of C4H4O4 and a melting point of 130-134°C.
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Maleic anhydride is a chemical compound used as a raw material in the production of various industrial and consumer products. It is a colorless, crystalline solid with a distinctive odor. Maleic anhydride serves as a building block in the synthesis of a wide range of chemicals, including resins, plasticizers, and agricultural products. Its core function is to provide a starting material for these diverse applications, without specific interpretation or extrapolation on its intended use.
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Sodium hydroxide is a chemical compound with the formula NaOH. It is a white, odorless, crystalline solid that is highly soluble in water and is a strong base. It is commonly used in various laboratory applications as a reagent.
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Hydrochloric acid is a commonly used laboratory reagent. It is a clear, colorless, and highly corrosive liquid with a pungent odor. Hydrochloric acid is an aqueous solution of hydrogen chloride gas.
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Acetic acid is a colorless, vinegar-like liquid chemical compound. It is a commonly used laboratory reagent with the molecular formula CH3COOH. Acetic acid serves as a solvent, a pH adjuster, and a reactant in various chemical processes.
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Citric acid is a commonly used chemical compound in laboratory settings. It is a weak organic acid that can be found naturally in citrus fruits. Citric acid has a wide range of applications in various laboratory procedures and analyses.
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Blocking reagent is a laboratory product used to prevent non-specific binding in various immunoassay techniques. It helps reduce background signals and improve the specificity of target analyte detection.
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Methanol is a clear, colorless, and flammable liquid that is widely used in various industrial and laboratory applications. It serves as a solvent, fuel, and chemical intermediate. Methanol has a simple chemical formula of CH3OH and a boiling point of 64.7°C. It is a versatile compound that is widely used in the production of other chemicals, as well as in the fuel industry.
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Oleic acid is a long-chain monounsaturated fatty acid commonly used in various laboratory applications. It is a colorless to light-yellow liquid with a characteristic odor. Oleic acid is widely utilized as a component in various laboratory reagents and formulations, often serving as a surfactant or emulsifier.
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Chloroform is a colorless, volatile liquid with a characteristic sweet odor. It is a commonly used solvent in a variety of laboratory applications, including extraction, purification, and sample preparation processes. Chloroform has a high density and is immiscible with water, making it a useful solvent for a range of organic compounds.
More about "Maleic acid"
Maleic acid, also known as cis-butenedioic acid, is a dicarboxylic acid with the chemical formula C4H4O4.
It is a white, crystalline solid that is commonly used in the production of various chemicals and materials.
Maleic acid has a wide range of applications, including the synthesis of resins, plasticizers, and pharmaceuticals.
It is also used in the production of maleic anhydride, which is a precursor to many important industrial chemicals.
Maleic acid is an important intermediate in the chemical industry and has been the focus of extensive research and development efforts.
It can be synthesized through the oxidation of n-butane or the hydrolysis of maleic anhydride.
Maleic acid is often used in conjunction with other compounds, such as sodium hydroxide, hydrochloric acid, acetic acid, citric acid, blocking reagents, methanol, oleic acid, and chloroform, to create a wide variety of products.
One of the key applications of maleic acid is in the production of resins, which are used in a variety of industries, including construction, automotive, and electronics.
Maleic acid is also used as a plasticizer, which helps to make materials more flexible and durable.
In the pharmaceutical industry, maleic acid is used in the synthesis of various drugs and medicinal compounds.
Overall, maleic acid is a versatile and important chemical that has a wide range of applications in both the industrial and medical fields.
Its unique properties and diverse uses make it an essential component in the production of many everyday products.
It is a white, crystalline solid that is commonly used in the production of various chemicals and materials.
Maleic acid has a wide range of applications, including the synthesis of resins, plasticizers, and pharmaceuticals.
It is also used in the production of maleic anhydride, which is a precursor to many important industrial chemicals.
Maleic acid is an important intermediate in the chemical industry and has been the focus of extensive research and development efforts.
It can be synthesized through the oxidation of n-butane or the hydrolysis of maleic anhydride.
Maleic acid is often used in conjunction with other compounds, such as sodium hydroxide, hydrochloric acid, acetic acid, citric acid, blocking reagents, methanol, oleic acid, and chloroform, to create a wide variety of products.
One of the key applications of maleic acid is in the production of resins, which are used in a variety of industries, including construction, automotive, and electronics.
Maleic acid is also used as a plasticizer, which helps to make materials more flexible and durable.
In the pharmaceutical industry, maleic acid is used in the synthesis of various drugs and medicinal compounds.
Overall, maleic acid is a versatile and important chemical that has a wide range of applications in both the industrial and medical fields.
Its unique properties and diverse uses make it an essential component in the production of many everyday products.