For the mineralization solution (Sigma-Aldrich, Steinheim, Germany, purity) was used ( , and ). As solvent a mixture of ( ) and ethanol (Roth, Karlsruhe, Germany, ≥99.8%, p.a., purity) with 80 vol.% ethanol was chosen. For zetapotential measurements of mineralized particles in sodium chloride (Merck, Darmstadt, Germany) the pH value was adjusted using hydrochloric acid (Roth, fuming ) and sodium hydroxide (Merck).
Ethanol
Manufactured by Carl Roth
Sourced in Germany, Austria, United States
Ethanol is a colorless, volatile liquid with a characteristic odor. It is a flammable chemical compound commonly used in various laboratory applications. Ethanol serves as a solvent, disinfectant, and reagent in various scientific and research processes.
Automatically generated - may contain errors
Lab products found in correlation
Methanol, by Carl Roth (12 mentions)
Hydrochloric acid (hcl), by Carl Roth (8 mentions)
Acetone, by Carl Roth (6 mentions)
Potassium chloride (kcl), by Carl Roth (5 mentions)
Toluene, by Merck Group (5 mentions)
Sodium chloride (nacl), by Carl Roth (4 mentions)
Acetic acid, by Carl Roth (4 mentions)
2 propanol, by Carl Roth (4 mentions)
Acetonitrile, by Carl Roth (4 mentions)
Formic acid, by Merck Group (4 mentions)
Sodium hydroxide (naoh), by Carl Roth (4 mentions)
Hydrochloric acid (hcl), by Merck Group (4 mentions)
Acetonitrile, by Avantor (3 mentions)
Potassium dihydrogen phosphate, by Carl Roth (3 mentions)
Methanol, by Avantor (3 mentions)
Ethyl acetate, by Carl Roth (3 mentions)
Tetrahydrofuran, by Carl Roth (3 mentions)
Chloroform, by Carl Roth (3 mentions)
α cyano 4 hydroxycinnamic acid, by Merck Group (3 mentions)
Biotyper 3, by Bruker (3 mentions)
97 protocols using ethanol
1
Zirconium Oxide Mineralization Protocol
2
MALDI-MSI Tissue Staining and Imaging
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After MALDI MSI, the 9-AA matrix was removed from the tissue section surface using 70% ethanol (Carl Roth) for 4 minutes, followed by H&E staining of the very same tissue sections. The H&E-stained tissue sections were cover-slipped and scanned with an AxioScan.Z1 digital slide scanner (Carl Zeiss) equipped with a 20× magnification objective. The visualization and export of the images to TIFF was done with the software ZEN 2.3 blue edition (Carl Zeiss).
3
Quantification of Extracellular Calcium Deposition
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To quantify the amount of extracellular calcium deposition, the cells were subjected to Alizarin Red S staining as published previously [26 (link),27 (link)]. In short, cells were fixed in 70% ethanol (Carl Roth, Karlsruhe, Germany), incubated over night at 4 °C, washed with Aqua dest. and then stained with 0.5% Alizarin Red S solution (Waldeck, Münster, Germany) for 10 min. After washing with PBS, a 10% hexadecylpyridinium chloride solution (Merck, Darmstadt, Germany) was added to each sample and incubated on an oscillator (IKA-Werke, Staufen, Germany) for 30 min at 350 rpm to dissolve the stained calcium depositions. After complete dissolution each sample was measured spectrometrically at 570 nm as technical duplicates and normalized to a standard curve.
4
Automated RNA Extraction using KingFisher
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The KingFisher™ (KF) Duo system was applied with a 12 pin magnet head which enabled processing of 12 samples per run using microtiter 96 deepwell plates (Thermo Fisher). Prior to the extraction process, the deepwell plates were filled with the following reagents: lysis buffer VXL, MagAttract Suspension B, buffer AW1, buffer AW2 (Qiagen), 100% Ethanol (Carl Roth GmbH, Karlsruhe, Germany), nuclease-free water and buffer AVE (Qiagen) [see Additional file 1 : Table S1]. For extraction, 100 μl of the sample were added to the lysis buffer and mixed by pipetting. After that, binding buffer ACB was added and automated extraction was executed and completed within 8 min using the protocol indicated in Table 1 . The extracted RNA was eluted in 100 μl and subsequently transferred to 1.5 ml tubes for storage.
5
Metabolite Extraction from Aortic Tissue
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Following surgical extraction, cleaning, and rinsing of ascending thoracic aortic tissue samples, samples were immediately snap frozen in liquid nitrogen and stored at -80°C until further use. For metabolite extraction, per sample 40 mg of aortic tissue were placed in sterile polystyrene tube on dry ice, followed by adding 6 μl ice cold 100% ethanol (ultrapure; Carl Roth, Karlsruhe, Germany) per mg of tissue. Consequently, a 5 mm diameter hardened steel ball (Retsch, Haan, Germany) was added into the tubes, and tubes were placed in a Mixer Mill MM400 (Retsch, Haan, Germany), followed by breaking up of tissue pieces by applying a vibrational frequency of 30 Hz for 3 minutes. In the next step samples were sonicated (15 intervals, cycles: 10%, power: MS72/D; Sonopuls HD 200, Bandelin, Berlin, Germany) and by a centrifugation step (10 min/20,800 x g/4°C) all solid components were separated. Supernatants were collected and stored on dry ice until metabolite analyses.
6
Ethanolic Extraction of Plant Compounds
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Ethanolic extracts were prepared from the roots and rhizomes of the plant. Sliced dried plant material was ground and about 50 g was used for the extraction. The lipophilic compounds were removed by pre-extraction with hexane (Carl Roth, Karlsruhe, Germany). Ethanolic extraction with 96% ethanol (Carl Roth, Karlsruhe, Germany) took place in an ultrasonic bath (Elma, Singen, Germany) for 10 min at room temperature, followed by centrifugation at 4000 RPM for 10 min. Ethanolic extraction was repeated three times, the supernatants were collected, and the solvent was evaporated using a rotary evaporator (Heidolph, Schwabach, Germany), followed by freeze drying using a VirTis Sentury freeze dryer (SP Scientific, Buena, CA, USA) resulting in 28.52% (“Mattmark”) and 32.47% (“Rosavine”) extraction yields. The ethanolic extracts, designated as M/R 96% EtOH, were stored in dark glass flasks in a fridge at 4 °C.
7
Quantification of Sal-complex in BGs
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In order to determine the amount of Sal-complex within the BGs, 4 mg loaded BGs were resuspended in 0.5 mL of 96% ethanol (Carl Roth, Vienna, Austria), followed by 5 min of ultrasonication. Subsequently, the ethanolic extract was diluted equally with Milli-Q® H2O (1:1) and immediately centrifuged at 11,300× g for 15 min at 4 °C. FPLC analysis was performed using an AKTÄ Purifier 10 System® (GE Healthcare, Chicago, IL, USA) equipped with a Superdex 10/300 GL column (24 mL; Cytiva, Germany), and UV and conductivity detectors. MeOH-acetate (65:35) was used as an eluent. The quantification was carried out using the peak area method applying free Sal-Mn(II) complex as an external standard.
8
Stöber Synthesis of Silica and Silica/polyP Nanoparticles
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Silica NP: Silica/polyP NP : The silica-polyP NP were similarly prepared. The water/ammonia solution (60 mL) was supplemented with 0.1 g of polyP3 or polyP40. Then ethanol (30 mL) was added. Finally, the reaction mixture was supplemented with 4 mL of TEOS then processed at 50 °C as above; silica/polyP-NP either as “silica/polyP3-NP” or “silica/polyP40-NP”, respectively.
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9
Extraction and Characterization of Bioactive Compounds from St. John's Wort
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Aerial parts (leaves, stem, petals, and flowers)
of H. perforatumL (St. John’s wort) were collected in July–August
2018 from the Ghab Plain in Syria (Google maps: 35.586856, 36.355724
and 180–200 m above sea level) and harvested during the flowering
season. Hypericin and quercetin were purchased from Cayman Pharma
(Neratovice, Czech Republic); hyperoside was purchased from Roth (Karlsruhe,
Germany); quercitrin hydrate, chlorogenic acid, 1,1-diphenyl-2-picrylhydrazyl
(DPPH), potassium persulfate, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS) were purchased from Sigma-Aldrich (Darmstadt, Germany);
Whatman 90 mm filter paper was purchased from GE Healthcare Life Sciences
(Freiburg Germany); a 0.22 μm nylon syringe filter, Sartolab
Vakuumfilter 180C5, 0.22 μm polyethersulfon, 500 mL, 25 mm syringe
filter, 0.45 μm RC with GF prefilter, and 0.45 μm PTFE
filter were purchased from Sartorius (Goettingen, Germany); and 0.45
μm prefilter was purchased from Wicom (Heppenheim, Germany).
Ethanol, mEthanol, and acetone were HPLC grade from Roth (Karlsruhe,
Germany); acetonitrile was obtained from VWR (Hannover, Germany),
and water was purified using a QM system from Sartorius (Goettingen,
Germany).
of H. perforatumL (St. John’s wort) were collected in July–August
2018 from the Ghab Plain in Syria (Google maps: 35.586856, 36.355724
and 180–200 m above sea level) and harvested during the flowering
season. Hypericin and quercetin were purchased from Cayman Pharma
(Neratovice, Czech Republic); hyperoside was purchased from Roth (Karlsruhe,
Germany); quercitrin hydrate, chlorogenic acid, 1,1-diphenyl-2-picrylhydrazyl
(DPPH), potassium persulfate, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS) were purchased from Sigma-Aldrich (Darmstadt, Germany);
Whatman 90 mm filter paper was purchased from GE Healthcare Life Sciences
(Freiburg Germany); a 0.22 μm nylon syringe filter, Sartolab
Vakuumfilter 180C5, 0.22 μm polyethersulfon, 500 mL, 25 mm syringe
filter, 0.45 μm RC with GF prefilter, and 0.45 μm PTFE
filter were purchased from Sartorius (Goettingen, Germany); and 0.45
μm prefilter was purchased from Wicom (Heppenheim, Germany).
Ethanol, mEthanol, and acetone were HPLC grade from Roth (Karlsruhe,
Germany); acetonitrile was obtained from VWR (Hannover, Germany),
and water was purified using a QM system from Sartorius (Goettingen,
Germany).
10
Oxidative Stress and Lipid Peroxidation Assay
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2,4-Dinitrophenyl hydrazine (DNPH), paraquat (PQ), thiourea, 7-(diethylamino)-coumarin-3-carbohydrazide (CHH), Hoechst 33,258 (Hoechst), 2,7-dichlorofluorescin diacetate (DCFDA), tert-butyl methyl ether (MTBE), primary goat anti-DNP Ab and sodium, potassium, and ammonium salts were purchased from Sigma Aldrich GmbH (Taufkirchen, Germany). Formic acid was obtained from BiosolveBV (Valkenswaard, Netherlands). Dithiothreitol (DTT), urea, and ethanol were obtained from CarlRoth GmbH & Co. KG and chloroform was from Merck KGaA (Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM/Ham's F12), fetal bovine serum (FBS), phosphate buffer saline (PBS), 7-aminoactinomysin-D (7-AAD), Trypan Blue (0.1%) and antibiotic (penicillin/streptomycin) solutions were obtained from Life Technologies GmbH (Darmstadt, Germany). Secondary rhodamine (TRITC) AffiniPure Rabbit Anti-Goat IgG (H+L) Ab and peroxidase-conjugated donkey anti-goat Abs were obtained from Jackson ImmunoResearch Laboratories, Inc. (Pennsylvania, United States). E06-monoclonalAb-TopFlour™ antibody was purchased from Avanti Polar Lipids, Inc. (Alabama, United States of America). Low fluorescent PVDF membranes, immunoblot blocking solution (AdvanBlock), immunoblot washing solution (AdvanWash) were purchased from Advansta (California, United States of America).
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