Samples were treated with Oil Red O (Sigma, O0625) staining or Nile Red (Sigma, 19123) staining to detect LDs. For Oil Red O staining, samples were washed in 60% isopropanol for 5 min and then incubated with Oil Red O working solution (300 mg Oil Red O in 100 ml isopropanol, and 6:4 diluted with water) for 20 min at room temperature. After washing with 60% isopropanol for 1 min and H2O for 5 min, the slides were mounted with DAPI Fluoromount-G (SouthernBiotech, 0100-20) for imaging. For Nile Red Staining, slides were stained with Nile Red (Sigma, 19123) at 1 μg ml−1 for 10 min at room temperature, washed with PBS and mounted with DAPI Fluoromount-G (SouthernBiotech, 0100-20).
Nile red
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, China, Italy, France, Sao Tome and Principe, Macao, Canada, Israel, Switzerland, Ireland, Spain, Australia, Japan, India
Nile Red is a fluorescent dye used in laboratory settings. It is a lipophilic stain that selectively stains neutral lipids, making it a useful tool for the detection and quantification of lipids in various samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (41 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (23 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (22 mentions)
Hoechst 33342, by Merck Group (21 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (20 mentions)
Nile blue, by Merck Group (18 mentions)
Oil red o, by Merck Group (18 mentions)
Bovine serum albumin (bsa), by Merck Group (17 mentions)
Hoechst 33342, by Thermo Fisher Scientific (17 mentions)
Dexamethasone (dex), by Merck Group (16 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (15 mentions)
Hydrochloric acid (hcl), by Merck Group (15 mentions)
Methanol, by Merck Group (15 mentions)
Fast green fcf, by Merck Group (13 mentions)
Insulin, by Merck Group (13 mentions)
Rhodamine b, by Merck Group (12 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (12 mentions)
Nile blue a, by Merck Group (11 mentions)
Sodium hydroxide (naoh), by Merck Group (11 mentions)
Lsm 710, by Zeiss (10 mentions)
853 protocols using nile red
1
Lipid Droplet Staining Protocols
2
Confocal Microscopy Evaluation of Cheese Microstructure
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Confocal laser scanning microscopy (CLSM) was applied to study the microstructure of the DC and NDC. Sampling was carried out as previously performed by D’Incecco and co-workers (2020) with some modifications [28 (link)]. Three portions of cheese (2 × 2 × 1 mm) were cut using a razor blade from each cheese slice at a 5 cm depth from the rind. Additional sampling was performed close to the defect for the DC. The cheese portions were stained within embryo dishes (Electron Microscopy Sciences, Hatfield, PA, USA) using Nile Red (Sigma Aldrich, St. Louis, MO, USA) to visualise the fat and Fast Green FCF (Sigma Aldrich) to visualise the protein. Just before staining, both stock solutions of Nile Red (1 mg/mL in dimethyl sulfoxide) and Fast Green (1 mg/mL in Millipore MilliQ water) were diluted tenfold in water. The samples were analysed using an inverted confocal laser scanning microscope, A1+ (Nikon, Minato, Japan). The Nile Red was excited at 488 nm and the emission was collected at 520–590 nm. The Fast Green was excited at 638 nm and the emission was collected at 660–740 nm.
3
CLSM Analysis of Cheese Microstructure
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Microstructure of cheese samples was analyzed by Confocal Laser Scanning Microscopy (CLSM) as described previously [16 (link)]. Three portions of cheese (around 2 mm × 2 mm ×1 mm) were taken from each cheese slice at 5-cm depth using a razor blade. Samples were stained with Nile Red (Sigma Aldrich, St Louis, MO, USA) and Fast Green FCF (Sigma Aldrich) to visualize fat and protein matrix, respectively. The staining was carried out as follows: the stock solutions of Nile Red (1 mg/mL in dimethyl sulfoxide) and Fast Green (1 mg/mL in Millipore MilliQ purified water) were diluted tenfold just prior to 5-min staining. Samples were analyzed by using an inverted confocal laser scanning microscope A1+ (Nikon, Minato, Japan). The excitation/emission wavelengths were set at 488 nm/520–590 nm for Nile Red and at 638 nm/660–740 nm for Fast Green FCF [17 (link)]. Images are presented as maximum projection of 23 optical sections stacked together with separation between layers set at 0.30 μm. Image analysis was performed using Vision4D software (Arivis, AG, Germany) on maximum projection of CLSM z-stack images. Porosity was calculated as the ratio between the nonfluorescent volume (µm3) and the total fluorescent volume (µm3).
4
Intracellular Lipid Quantification by Flow Cytometry
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Intracellular lipid content was determined by flow cytometry using lipophilic dye Nile red. Aliquots were collected after 0, 6, 10, 12 and 24 h of incubation with 9 μg/mL of argentilactone and in the absence of the compound. The cells were washed twice with PBS and incubated with 2 μg/mL Nile red (Sigma Aldrich), for 15 min at room temperature. Nile red intracellular fluorescence was determined by guava easyCyte™ Flow Cytometers (Merck Millipore, Billerica, EUA) on emission channel of 585 nm and excitation 488 nm. A total of 5000 cells were collected to analysis.
5
Oil-in-Water Emulsion Formation and Characterization
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Oil droplets are made from soybean oil (Sigma-Aldrich, St. Louis, MO, USA). Briefly, soybean oil was dispersed and emulsified by hand in an aqueous continuous phase containing 15% w/w of Poloxamer 188 block polymer surfactant (CRODA, East Yorkshire, UK) and 1% w/w sodium alginate (Sigma-Aldrich, St. Louis, MO, USA) at a final oil fraction equal to 75%. The rough emulsion was sheared in a Couette cell apparatus at a controlled shear rate of 110 rpm as described by Mason et al.39 (link). For storage and handling purposes the emulsion are diluted to an oil fraction of 60% w/w with 1% w/w of poloxamer 188 in the continuous phase and stored at 12 °C in a Peltier-cooled cabinet.
To stain droplets with Nile Red (Sigma-Aldrich, St. Louis, MO, USA), a red lipophilic dye, the droplets suspension is washed and resuspended in cell growth complete media containing 10 μM of Nile Red. Size distribution of the emulsion droplets was measured by brightfield microscopy and image analysis.
To stain droplets with Nile Red (Sigma-Aldrich, St. Louis, MO, USA), a red lipophilic dye, the droplets suspension is washed and resuspended in cell growth complete media containing 10 μM of Nile Red. Size distribution of the emulsion droplets was measured by brightfield microscopy and image analysis.
6
Nile Red Staining of Pupae
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Nile red (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in acetone (500 μg/ml) and stored at 4 °C under darkness to prepare the stock solution for Nile red staining. Approximately 10 μl of stock solution was added to 10 ml of 75% glycerol. The Nile red staining solution was applied to pupae for 5–10 min to stain the respiratory trumpets of pupae7 (link). After staining, pupae were washed with fresh water to avoid any non-specific binding of Nile dyes in their body. The stained pupae were examined using a fluorescent microscope (Nikon, Tokyo, Japan).
7
Visualizing Pathogen Development in Plant Calli
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During tissue culture, Nile red (Sigma‐Aldrich, Oakville, Canada) and calcofluor white (CFW, Sigma‐Aldrich, Oakville, Canada) double‐staining techniques were used to observe pathogen development in calli. Calli were incubated in a final solution of 1 µg/ml Nile red, diluted in sterile water from a 10 mg/ml stock, for 10 min in the dark. After a wash with sterile water, calli were incubated in two drops of CFW for 5 min in the dark following the supplier's instruction. After a final wash, calli were mounted on glass slides for observations.
For live cell imaging of P. brassicae–Arabidopsis interactions, 4,6‐Diamidine‐2‐phenylindole dihydrochloride (DAPI, Sigma‐Aldrich, Oakville, Canada) staining of inoculated Arabidopsis roots was carried out in a working solution of 1 μg/ml DAPI, diluted from a 10 mg/ml stock in water, for 20 min in the dark, followed by a wash with sterile water and where appropriate, 1 µg/ml Nile red was added for 5 min. Images of stained roots were viewed and captured with a confocal laser‐scanning microscope (Zeiss Meta 510).
For live cell imaging of P. brassicae–Arabidopsis interactions, 4,6‐Diamidine‐2‐phenylindole dihydrochloride (DAPI, Sigma‐Aldrich, Oakville, Canada) staining of inoculated Arabidopsis roots was carried out in a working solution of 1 μg/ml DAPI, diluted from a 10 mg/ml stock in water, for 20 min in the dark, followed by a wash with sterile water and where appropriate, 1 µg/ml Nile red was added for 5 min. Images of stained roots were viewed and captured with a confocal laser‐scanning microscope (Zeiss Meta 510).
8
Quantitative Analysis of Sebaceous Lipids
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To detect sebaceous lipids, human sebocytes were seeded in 12-well culture plates at a density of 1 × 105 cells per well and cultured overnight, after which they were treated every two days with CTSE or CP for a total of four days. For Nile Red staining, a stock solution of Nile Red (Sigma-Aldrich, St. Louis, MO, USA; 1 mg/mL in acetone) was diluted to a final concentration of 10 μg/mL in phosphate-buffered saline (PBS). Cells were fixed in 4% formaldehyde at room temperature for 10 min, stained with Nile Red solution for 15 min at 37 °C, and washed with PBS. Stained cells were visualized by fluorescence microscopy using 485-nm excitation and 565-nm emission filters. Lipids were quantitatively measured using Leica Application Suite (LAS) X software.
9
Quantifying Lipid Accumulation in HepG2 Cells
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HepG2 cells were incubated in black 96-well plates (Cat# 655090; Greiner) at a density of 4 × 105 cells per well and treated with O:P (2:1, respectively) at final concentrations of 0.3–1 mM for 24 h. Each well was washed twice with 1x PBS and then stained with Nile Red (Cat# 19123; Sigma–Aldrich) and Hoechst by adding a Nile Red/Hoechst mixed solution (1 μg/mL; diluted in 1x PBS) to the cells for 15 min at 37 °C. The cells were then washed with 1x PBS, and the fluorescence intensity was measured with a SpectraMax iD3 microplate reader (Molecular Devices) at wavelengths of ex:488/em:550 and ex:350/em:461 nm for Nile Red and Hoechst, respectively. The results were normalized to the total protein levels of each well and presented as a change in the accumulation of lipids in comparison with the Veh-treated group.
10
Fluorescent Staining Techniques for Fungal Analysis
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The DAB (3,3-diaminobenzidine, Sigma-Aldrich, USA) solution with a concentration of 0.1 mg/ml was used to detect ROS in host cells infected by different strains. For CFW staining, mycelia and conidia were harvested from CM medium and stained with 10 μg/ml CFW (Sigma-Aldrich, United States) for 10 min in the dark, then observed under the Nikon microscope Ni90 (Nikon, Tokyo, Japan). Glycogen was detected by KI/I2 staining solution (60 mg/ml KI and 10 mg/ml I2) (Thines et al., 2000 (link)). Lipid droplets were stained using Nile Red solution, consisting of 50 mM Tris/maleate buffer (pH 7.5), 20 mg/ml polyvinylpyrrolidone, and 2.5 mg/ml Nile Red (Sigma-Aldrich, St. Louis, MO, USA). For different kinds of staining assays, the spore suspension (5 × 105 spores/ml) was dropped on plastic coverslips and kept in growth chamber and stained for 15 min at different time points, then detected using an epifluorescence microscope (Ni90, Nikon, Tokyo, Japan).
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