We visualized mucin by labelling it with an Atto590 N-hydrosuccinimide ester dye (λex\λem = 594/624 nm, Sigma-Aldrich®). Briefly, we added 100 mg of mucin in 20 ml phosphate buffer (100 mM pH 8.5) to 1 mg of the dye dissolved in amine-free dimethylformamide (Molecular Biology Grade, Sigma-Aldrich®). We continually stirred the solution for 2 h at 22°C. Then, we added nine parts by volume of previously cooled aqueous ethanol solution (90% v/v) at −80°C to the suspension to induce precipitation of the mucin. We precipitated mucin overnight by maintaining the temperature of the suspension at −20°C. We recovered the precipitate by centrifuging the suspension at 50 000g for 15 min. We then washed the precipitate with cold ethanol (−80°C) to remove as much of the residual Atto590 dye, which is highly soluble in ethanol, as possible. We resuspended the dried mucin in 20 ml nanopure water and dialysed it overnight to remove phosphate ions that originated from the buffer. To visualize DPPC, we added 100 µg boron-dipyrromethene-phosphocholine (BODIPY-PC, λex\λem = 488\503 nm, Molecular Probes, Life Technologies Inc.) or 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC, λex\λem = 460\534 nm, Avanti Polar Lipids Inc.) to unlabelled DPPC.
Molecular biology grade
Manufactured by Merck Group
Sourced in United States
Molecular biology grade lab equipment from Merck Group is designed for use in molecular biology applications. These products meet high standards of quality and purity to support reliable and reproducible results in research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bodipy pc, by Thermo Fisher Scientific (1 mentions)
Agarose, by Merck Group (1 mentions)
4 aminoantipyrine, by Merck Group (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Histidine, by Merck Group (1 mentions)
Salicylaldehyde, by Merck Group (1 mentions)
Leica bond rx, by Leica camera (1 mentions)
Mr frosty freezing container, by Thermo Fisher Scientific (1 mentions)
Quant it picogreen dsdna assay, by Thermo Fisher Scientific (1 mentions)
Fluostar omega microplate reader, by BMG Labtech (1 mentions)
7 protocols using molecular biology grade
1
Fluorescent Labeling of Mucin and DPPC
2
Synthesis and Characterization of Metal Complexes
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4-aminoantipyrine, Salicylaldehyde, Histidine and all metal chloride salts were procured from Merck (Darmstadt, Germany). Solvents used for electrochemical and spectroscopic studies have been purified by standard [27] (link) procedures. Himedia chemicals were used as such for antimicrobial studies. CT- DNA was purchased from Genei Bangalore (India). Ethidium bromide (EB) and Agarose (molecular biology grade) were obtained from Sigma–Aldrich (St. Louis, Missouri, USA). Tris (hydroxymethyl)aminomethane hydrochloride (Tris–HCl) buffer solution was prepared using deionized and distilled water.
3
Anatomical Mapping of Brain Regions
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The brain was dissected on a stereotaxic frame to obtain sagital and corona sections of the MCA, periventricular zone (PVZ) and parietal cortex (PC) using specific coordinates [2.5 mm lateral to the median fissure (sagital) and 1.5mm anterior to the bregma (corona section)]. Later, the sections were post-fixed in formolcalcium in a separate chamber (6 hours) and processed into paraffin wax embedded tissue blocks. Leica Bond RX (Leica GmBH, Germany) automated tissue stainer was used for immunohistochemical processing. All antibodies were procured from Novocastra (Leica Biosystems, Germany). Reagents and buffers used were molecular biology grade (99.9% pure) from Sigma-Aldrich (Germany).
4
Innaxon LPS and Sigma LTA Protocols
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Reagents and chemicals were molecular biology grade from Sigma (St. Louis, MO, USA) or Life Technologies (Carlsbad, CA, USA). Ultrapure LPS from E. coli 055:B5 (S-form; purity ≥ 99.9%, 1 ng = 10 endotoxin units) was from Innaxon (Oakield Close, UK). LTA from E. faecalis was from Sigma (St. Louis, MO, USA).
5
Lyophilized Nanocarrier Formulations
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The mixtures of NCs (3 mg/mL), HA (1.5 mg/mL), and cryoprotectants (25 mg/mL) were prepared and frozen using either a fast or slow freezing process before lyophilization. The weight percentage of NCs in the lyophilized formulation was about 10.2%. For the fast-freezing process, the mixture was frozen rapidly using liquid nitrogen and then dried using a lyophilizer. For the slow-freezing process, the mixture was frozen using a Mr. Frosty freezing container at a cooling rate of −1 °C min−1 (Thermo Fisher Scientific) and then dried using a lyophilizer. Lyophilized samples were stored at 4 °C and reconstituted simply by water (molecular biology grade, Sigma).
6
ASFV p72 gene genotypes protocol
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Synthetic double-stranded DNAs for different genotypes of ASFV p72 gene were used for analytical performance tests of the assay. Twenty-four genotypes of ASFV p72 gene were classified into seven genotype groups based on nucleic acid sequences of the p72 target region (Table 1 ). The seven genotype groups were designated as GT2, GT3, GT7, GT8, GT9, GT10 and GT23, referring to the representative genotype of each group. A double-stranded DNA of the p72 conservative region with a size of about 400 bp (gBlocks, IDT, Singapore) was synthesized for each genotype group and used as a standard sample. The synthesized DNAs were dissolved in nuclease-free water (Molecular Biology grade, Sigma-Aldrich, USA) to the concentration of 1 × 109 copies/μL, aliquoted and stored at −70°C.
7
Quantifying DNA Content in Cell-Laden Hydrogels
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Quant-iT PicoGreen dsDNA assay (Invitrogen) was used to quantify the DNA content. Constructs on days 1, 7, and 21 were washed with DPBS, moved to fresh well plates, and then frozen at -80 °C. The well plates, where the constructs were cultured, were also frozen for further analysis. Upon sample thawing, 1.5 mL of a digestion cocktail consisting of 100 μg mL -1 of proteinase K (Meridian Bioscience) and 0.1% Triton X-100 in nuclease-free water, molecular biology grade (Sigma-Aldrich), was added per each well. Samples were incubated overnight at 37 °C to digest hydrogel matrices and lyse cells. Then the sample lysate was diluted at 1:10 in Tris-EDTA (TE) buffer. 100 μL of the diluted sample lysate was mixed with 100 μL of PicoGreen solution, incubated for 10 min in the dark, and measured (excitation and emission filters of 485 and 520 nm, respectively) in a 96-well plate using a FLUOstar Omega microplate reader (BMG Labtech, Germany). DNA standards were prepared according to the manufacturer's protocol. Three samples per time point were evaluated (n = 3), and each sample was measured in triplicates.
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