Zebrafish hearts were extracted and fixed in 4% (wt/vol) paraformaldehyde (Alfa Aesar, MA) at room temperature for 1 hr. Collected hearts were subsequently cryopreserved with 30% (wt/vol) sucrose, followed by immersed in OCT (Tissue-Tek, Sakura Finetek, Torrance, CA) and stored at −80°C immediately. 10 μm cryosections were collected for histological analysis. AFOG staining was applied for the visualization of healthy CMs in orange, collagens in blue and fibrins in red. In brief, samples were incubated in preheated Bouin’s solution (Sigma) at 58°C for 2 hr post fixation and subsequently immersed in 1% phosphomolybdic acid (Sigma) and 0.5% phosphotungstic acid solution (Sigma) at room temperature for 5 min for mordanting. Samples were then incubated with AFOG solution containing Aniline Blue (Sigma), Orange G (Sigma), and Acid Fuchsin (Sigma) for color development. Quantification was done by measuring the scar area in each heart from five discontinuous sections including the one with the largest scar as well as the two sections at the front and the two sections at the back as previously described (Lai et al., 2017 (link)). Statistic was performed on Prism 9 using Student’s t-test.
Phosphotungstic acid solution
Manufactured by Merck Group
Sourced in United States
Phosphotungstic acid solution is a chemical reagent used in various laboratory applications. It is a clear, colorless liquid that serves as a precipitating agent for certain proteins and nucleic acids. The primary function of this solution is to facilitate the separation and isolation of these biomolecules during analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Acid fuchsin, by Merck Group (1 mentions)
Aniline blue, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Preheated bouin s solution, by Merck Group (1 mentions)
Orange g, by Merck Group (1 mentions)
Phosphomolybdic acid, by Merck Group (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Potassium chloride (kcl), by Avantor (1 mentions)
Ethyl alcohol, by Avantor (1 mentions)
Acetonitrile, by Avantor (1 mentions)
N octanol, by Avantor (1 mentions)
Hydrochloric acid 37, by Scharlab (1 mentions)
Sodium phosphate monobasic, by Merck Group (1 mentions)
Potassium phosphate monobasic, by Merck Group (1 mentions)
Phospholipon 90g, by Lipoid (1 mentions)
Boric acid, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Scharlab (1 mentions)
Hydrogen peroxide 30, by Merck Group (1 mentions)
Tris hydroxymethyl aminomethane hydrochloride tris hcl, by Merck Group (1 mentions)
6 protocols using phosphotungstic acid solution
1
Zebrafish Heart Histological Analysis
2
Magnolol and Honokiol Extraction Protocol
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Magnolol and honokiol (98% purity) were acquired from New Natural Biotechnology (Shanghai, China). Phosphotungstic acid solution, sodium phosphate monobasic and potassium phosphate monobasic were purchased from Sigma-Aldrich (Madrid, Spain). N-octanol, acetonitrile, ethyl alcohol, and potassium chloride were obtained from VWR chemicals (Barcelona, Spain). Hydrochloric acid 37% and sodium hydroxide were purchased from Scharlab (Barcelona, Spain). Hydrogen peroxide 30% and boric acid were obtained from Merck (Barcelona, Spain). Phospholipon 90 G was provided by Lipoid GmbH (Ludwigshafen, Germany).
3
Synthesis of Magnetic Nanoparticles
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Iron(0)pentacarbonyl (Fe(CO)5, liquid 99.99%), 1-octadecene (ODE, technical grade 90%), oleic acid (OLEA, technical grade 90%), oleylamine (technical grade 70%), 1,2-dodecanediol (DDIOL, technical grade 90%), dopamine hydrochloride ((HO)2C6H3CH2CH2NH2HCl, powder), copper(II) sulfate pentahydrate (CuSO4•5H2O, powder), low metal trace 70% nitric acid and phosphotungstic acid solution (10% w/v) were purchased from Sigma-Aldrich and used as received. All solvents, namely chloroform, hexane, isopropanol and acetone, were also purchased from Sigma-Aldrich and were of the highest purity available. Phosphate-buffered saline (PBS), tris(hydroxymethyl) aminomethane hydrochloride (Tris-HCl) and borate buffers were prepared by using reagent-grade salts purchased from Sigma. All aqueous solutions were prepared by using water obtained from a Milli-Q Gradient A-10 system (18.2 MΩ cm, organic carbon content ≤ 4 µg L−1, Merck Millipore, Darmstadt, Germany).
4
Negative Staining of Extracellular Vesicles
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EV suspension (20 μl) was loaded onto formvar carbon‐coated copper electron microscopy grids for 2 min and then treated with phosphotungstic acid solution (12501‐23‐4, Sigma‐Aldrich Chemical Company, St Louis, MO, USA) for 5 min. Grids were washed three times with PBS to remove redundant phosphotungstic acid solution and then maintained in semi‐dry state. The images were observed under a TEM (Zeiss Inc., Thornwood, NY, USA).
5
Comprehensive Extracellular Vesicle Characterization
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Nanosight particle size analysis: 20 μg Evs were dissolved in 1 mL PBS and whirled for 1 min. Nanosight LM10 particle size analysis apprentice (NTA, Malvern instrument, UK) was used to observe the distribution of Evs.
Transmission electron microscope (TEM): 20 μL ultracentrifuged Evs were loaded into a carbon-coated microscope copper gridding for 2 min and then received negative staining with phosphotungstic acid solution (12,501-23-4, Sigma–Aldrich, USA) for 5 min. The gridding was washed in PBS three times to remove the excessive phosphotungstic acid solution and half-dried with filter paper. The Evs were observed under a Hitachi TEM (H7650, Japan) at 80 KV.
Western blot was applied to detect the expressions of biomarkers of Evs. The condensed Evs suspension was measured for protein concentration using a BCA kit (23,227, Thermofisher, USA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel was prepared for protein denature and electrophoresis. After the protein was transferred into the membrane, the expressions of CD9 (ab236630, abcam), CD63 (ab134045, abcam), CD81 (ab92726, abcam) and Calnexin (ab92573, abcam) were detected.
Transmission electron microscope (TEM): 20 μL ultracentrifuged Evs were loaded into a carbon-coated microscope copper gridding for 2 min and then received negative staining with phosphotungstic acid solution (12,501-23-4, Sigma–Aldrich, USA) for 5 min. The gridding was washed in PBS three times to remove the excessive phosphotungstic acid solution and half-dried with filter paper. The Evs were observed under a Hitachi TEM (H7650, Japan) at 80 KV.
Western blot was applied to detect the expressions of biomarkers of Evs. The condensed Evs suspension was measured for protein concentration using a BCA kit (23,227, Thermofisher, USA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel was prepared for protein denature and electrophoresis. After the protein was transferred into the membrane, the expressions of CD9 (ab236630, abcam), CD63 (ab134045, abcam), CD81 (ab92726, abcam) and Calnexin (ab92573, abcam) were detected.
6
Bacterial and Viral Sample Preparation for SEM and TEM Analysis
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Bacterial samples were harvested by centrifugation at 1500g for 5 min at 15 °C (HITACHI, RX2 series), and supernatants were removed. Then the bacteria were fixed overnight in the fixative containing 0.1 M phosphate-buffered solution (pH 7.3; Sigma) and 2% glutaraldehyde (Sigma) at 4 °C. Samples were then dehydrated with increasing concentrations of ethanol solutions (50%, 70%, 90%, and 100%; Sigma) before drying in 100% t-BuOH (Sigma) using a freeze-drying process (ilShin BioBase. TFD 8501). All the bacterial samples were dispersed on a metal grid in preparation for SEM characterization (JEOL, JSM-7500F). A total of 20 μL of the viral samples were pipetted on a TEM grid, then, after a 15 min air-drying process, samples were stained with 1% phosphotungstic acid solution (Sigma) for 1 min. The TEM grids were air-dried for TEM characterization (JEOL, JEM-2100F).
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