Sudan 3

Manufactured by Merck Group

Sourced in Germany, United States, Ireland, Japan

Sudan III is a dye used in laboratory applications. It is a powder-like substance that is soluble in organic solvents. Sudan III can be used as a staining agent for the detection and identification of lipids in biological samples.

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Sudan III staining (3 citations) Sudan III stain (2 citations)

41 protocols using sudan 3

Fabrication of Imogolite-Sudan III Composite Films

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The films of imogolite hydrogel, Sudan III, and both components on
SiO_x at ambient temperature were prepared
(Figure 2). Precipitation
of a 10 μL imogolite aliquot (drop) with a concentration of
12 mg/mL on the substrate was carried out by drop-casting. Preparation
of the imogolite-Sudan III (Sudan III-IMO) complex on the substrate
was carried out in two successive stages: deposition of a 10 μL
drop of a 0.1 mg/mL solution of Sudan III in tetrahydrofuran (THF)
(Merck) (deposited by spin-coating at 2000 rpm for 60 s on the substrate).
On the formed film, a 10 μL drop of a 12 mg/mL solution of imogolite
hydrogel was deposited by spin-coating at 2000 rpm for 60 s. The third
and last stage is deposition of a 10 μL drop of a 0.65 mg/mL
aqueous solution of Pluronic F68 (F68) on the substrate spin-coating
(it immediately) at 3200 rpm for 60 s.^{49 (link)} The scheme on the right represents the deposition of a 10 μL
drop of a 12 mg/mL aqueous solution of the Sudan III-imogolite (Sudan
III-IMO) complex containing 0.1 mg of Sudan III (Aldrich), which was
applied by drop-casting on the previously formed film (Figure 2).

Samith V.D., Navarro S, & Dabirian R. (2020). Morphological and Semi-empirical Study of the Pluronic F68/Imogolite/Sudan III Intersurfaces Composite for the Controlled Temperature Release of Hydrophobic Drugs. ACS Omega, 5(33), 20707-20723.

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Staining and Microscopy of Leaf Lipids

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Three days after treatment of the purified enzyme solution, leaves were cut into 1 cm × 1 cm fragments. The piece of leaf was held between slit of pith and sectioned by razor manually. The lipophilic layer of the leaf was stained with Sudan III (Sigma-Aldrich) according to Buda et al.^{42 (link)} with some modifications as follows: Leaf sections were soaked in the filtered Sudan III solution (2% w/v in 70% ethanol) for 8 min and were then rinsed first with 50% ethanol, followed by distilled water twice. Stained sections were mounted and observed under an ECLIPSE Ni light microscope with DS-Fi2 CCD camera system, photographed, and thickness of stained lipophilic layer was measured by NIS Elements ver. 4.40 software (Nikon, Tokyo, Japan).

Ueda H., Kurose D., Kugimiya S., Mitsuhara I., Yoshida S., Tabata J., Suzuki K, & Kitamoto H. (2018). Disease severity enhancement by an esterase from non-phytopathogenic yeast Pseudozyma antarctica and its potential as adjuvant for biocontrol agents. Scientific Reports, 8, 16455.

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Histological Lipid Staining in LAL Muscle

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This histological classical staining was performed in the LAL muscle of all the experimental groups and the controls at the end of the period of exposure (6 weeks). Sudan III stains lipids orange-red [27 (link)]. The LAL muscles were extracted and fixed in formalin.
The LAL muscle is a flat, thin muscle that does not require microtomy. Whole LAL muscles were immersed in the Sudan III preparation (50 mL of 50% alcohol, 50 mL acetone, 1 g Sudan III—Sigma-Aldrich, Steinheim, Germany) for 5 min. After cleaning the excess dye with 50% alcohol, a methylene blue contrast stain was performed (1 min). After washing off the excess under tap water, it was mounted on glycerin for visualization.

Gimenez-Donoso C., Bosque M., Vila A., Vilalta G, & Santafe M.M. (2020). Effects of a Fat-Rich Diet on the Spontaneous Release of Acetylcholine in the Neuromuscular Junction of Mice. Nutrients, 12(10), 3216.

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Preparation of Sudan III-Pluronic Micelles

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Nanopure water was used to prepare the micellar
solutions. A 32% (w/v) stock solution of Pluronic F68 (Aldrich) was
used to prepare the corresponding dispersions of Sudan III-F68 and
Sudan III-IMO-F68. Sudan III (Aldrich) was dissolved in a 10^–3 M concentration of a dispersion that contains 250 μL of THF,
and it was added to 500 μL of both dispersions, with constant
stirring in 1 mL tubes at 37 °C until complete evaporation of
the organic solvent.^{15 (link),29 (link)}

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Visualizing Lipid Accumulation in Cells

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Cells were seeded onto cover slips and fixed with 4% paraformaldehyde for 10 min at room temperature. Nuclei were stained with hemalum for 7 min and washed for 30 min. After incubation of slides in 50% ethanol for 15 sec, slides were stained with Sudan III (Merck Millipore, Darmstadt, Germany, 0.3 mg Sudan III in 70% Ethanol) for 15 min. To remove excessive stain slides were washed with 50% ethanol and then mounted on slides with Aquatex (Merck Millipore, Darmstadt, Germany). Lipid accumulation was observed under a Keyence BZ-9000 light microscope.

Hampel U., Schröder A., Mitchell T., Brown S., Snikeris P., Garreis F., Kunnen C., Willcox M, & Paulsen F. (2015). Serum-Induced Keratinization Processes in an Immortalized Human Meibomian Gland Epithelial Cell Line. PLoS ONE, 10(6), e0128096.

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Lipase and Pepsin Activity Assay

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Sodium hydroxide (NaOH), hydrochloric acid (HCl), trichloroacetic acid (TCA) and acetic acid were from Fisher Scientific (Dublin, Ireland). Kjeldahl tablets, sulfuric acid (>98%, H₂SO₄), boric acid, trifluoroacetic acid (TFA), Trizma^® base, dimethyl sulfoxide (DMSO), 2-mercaptoethanol, methanol, protein markers (6.5–200 kDa), Sudan III, 2,4,6-trinitrobenzenesulfonic acid (TNBS), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), porcine pepsin (2500 U/mg), porcine pancreatic lipase (50 mU/mL), 4-methylumbelliferyl oleate (4-MUO) and Orlistat^® were from Sigma-Aldrich (Dublin, Ireland). Hexane, MS-grade water and acetonitrile (ACN) were from Honeywell International Inc. (Dublin, Ireland). Comassie R, Laemmli buffer and Mini-Protean TGX 4–20% pre-cast polyacrylamide gels were from Bio-Rad Laboratories Inc. (Hercules, CA, USA). Sodium dodecyl sulfate (SDS) was from National Diagnostics (Atlanta, GA, USA). Corolase PP^® (4.4 U/mg) was from AB Enzymes GmbH (Darmstadt, Germany). Cellulose acetate filters were from Millipore (Carrigtwohill, Ireland) and polytetrafluoroethylene syringe filters were from VWR (Dublin, Ireland).

Khalesi M., Gcaza L, & FitzGerald R.J. (2023). In Vitro Digestibility, Biological Activity, and Physicochemical Characterization of Proteins Extracted from Conventionally and Organically Cultivated Hempseed (Cannabis sativa L.). Molecules, 28(3), 915.

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Raman Spectroscopy of Sudan III

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GERS measurements were performed using Renishaw inVia Raman microscope with 514 nm excitation and Sudan III as the target molecule. The samples were prepared for the measurements by depositing a 2 µl droplet of 0.1 mM Sudan III (analytical grade ≥96%, Sigma-Aldrich) diluted in acetone on the surface of the samples. The temperature dependence measurements were carried out using 0.25 mW power, 10 s integration time and 100X objective. In total, 9 measurements were recorded for each sample (600 °C, 700 °C, 800 °C and 900 °C) and the spectra were then averaged. The Raman mapping measurements were collected from a 200 µm × 600 µm area at 10 µm intervals using 0.5 mW power, 5 s integration time and 20X objective.

Kaplas T., Matikainen A., Nuutinen T., Suvanto S., Vahimaa P, & Svirko Y. (2017). Scalable fabrication of the graphitic substrates for graphene-enhanced Raman spectroscopy. Scientific Reports, 7, 8561.

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Staining Suberized Root Tissues

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Suberin in the root tissues was stained with alcoholic solutions of Sudan III (Sigma) [21 (link)]. Suberinized tissues were stained dark orange.

Akhiyarova G.R., Finkina E.I., Ovchinnikova T.V., Veselov D.S, & Kudoyarova G.R. (2019). Role of Pea LTPs and Abscisic Acid in Salt-Stressed Roots. Biomolecules, 10(1), 15.

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Azo Dye Degradation Assay

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Methyl Red, Sudan I, Sudan II, Sudan III, Sudan IV, Toluidine Red, Orange II, Fast Dark Blue R Salt, Lithol Rubin BK, Orange G, Amaranth, Ponceau BS, Direct Blue 15, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Inc. (Saint Louis, MO, USA). Solvent Red 1, Hansa Yellow, Hansa Orange, and Alphamine Red R were purchased from Fisher Scientific (Hanover Park, IL, USA). Diarylide Yellow HR (Pigment Yellow 83) was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Chemical structures of azo dyes used in this study were shown in Fig. 1. Tryptic Soy Agar (TSA) and Tryptic Soy Broth (TSB) used for bacterial growth were purchased from Fisher Scientific (Hanover Park, IL, USA). Stock solutions of azo dyes (10 mM) were prepared by using 100% DMSO as solvent, then dye solutions were diluted with TSB in degradation assay. Final concentration of DMSO and dye in the assays was 1% and 100 μM except as indicated otherwise.

Nho S.W., Cui X., Kweon O., Jin J., Chen H., Moon M.S., Kim S.J, & Cerniglia C.E. (2021). Phylogenetically diverse bacteria isolated from tattoo inks, an azo dye-rich environment, decolorize a wide range of azo dyes. Annals of microbiology, 71(1), 10.1186/s13213-021-01648-2.

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Azo Dye Degradation Assay

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