Oil red O (ORO) was purchased from Sigma, St. Louis, MO, USA. In brief, PHs were fixed with 4% paraformaldehyde for 30 minutes and then stained with 0.5% ORO (w:v) for 15 minutes at room temperature. All the stained sections were examined by light microscopy (400 × magnification). For Nile red (Sigma, St. Louis, MO, USA) staining, PHs were fixed as described previously and were co‐incubated with 0.1 mg/mL Nile red and 10 μg/mL DAPI for 10 minutes. After washing, Nile red‐stained neutral lipids (red) and DAPI‐stained nuclei (blue) were photographed with a Nikon fluorescence microscope (400 × magnification, ECLIPSE, Ts2R‐FL, Tokyo, Japan).
Nile red
Manufactured by Nikon
Sourced in Japan
Nile Red is a fluorescent dye used in laboratory applications. It is commonly used for the detection and quantification of neutral lipids, such as triglycerides and cholesterol esters, in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Nile red, by Merck Group (6 mentions)
Nile red, by Thermo Fisher Scientific (3 mentions)
Glomax multi detection system, by Promega (2 mentions)
Triolein, by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (2 mentions)
Eclipse ts2 microscope, by Nikon (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Nikon (2 mentions)
96 well clear bottom black tissue culture treated plates, by Corning (2 mentions)
Eclipse 80i, by Nikon (2 mentions)
Oil red o, by Merck Group (1 mentions)
Nucblue live readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Fluorescent microscope, by Nikon (1 mentions)
Coulter multisizer 4, by Beckman Coulter (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Eclipse ts100 filter g2a, by Nikon (1 mentions)
Milli q, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fast green fcf, by Merck Group (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
10 protocols using nile red
1
Lipid Visualization in Primary Hepatocytes
2
Fluorescent Imaging of E. coli in Beef
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Less than 1 g of 80% lean ground beef sample was placed on a glass slide. 10 μL solution of Nile red (Sigma-Aldrich; catalog no. 72485) dissolved in acetone (1 mg/mL) was added to the ground beef and the samples were lightly pressed down with coverslips. The samples were stained overnight in a refrigerator (4°C) before adding 20 μL of Hoechst-stained E. coli solutions. Nile red is very soluble in lipid and has an emission peak between 620–660 nm when excited in the range 525–575 nm. Each serial-diluted E. coli solution was stained with Hoechst dye that stains nucleic acids (NucBlue Live ReadyProbes Reagent, Life Technologies Corporation, Carlsbad, CA, USA; catalog no. R37605) for 30 minutes before it was applied to the Nile red-stained ground beef sample. Hoechst dye has a blue emission (around 460 nm) when excited in the ultraviolet (UV) range (around 350 nm). Once the Hoechst-stained E. coli solutions were applied to the Nile red-stained ground beef samples, they were firmly pressed down with the coverslips and were observed using a fluorescent microscope (Nikon, Tokyo, Japan). Images of each sample were taken under UV light and green light separately, and were overlayed using ImageJ (National Institutes of Health).
3
Quantifying Microalgal Lipid Content
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CrCUL RNAi transgenic strains and negative controls (pMaa7IR/X transgenic strains, shown as Maa7 lines) were grown under normal conditions (HSM medium) as described above for 10 days. The cell density was measured using a Coulter™ Multisizer 4 (Beckman Coulter, Fullerton, CA, USA) every day.
For neutral lipid content (NL) examination, cells were stained by 0.5 µg/mL of Nile red (Sigma-Aldrich, Saint Louis, MO, USA) dissolved in 25% (v/v) of DMSO solution. The fluorescence intensity (FI) was measured with a Glomax-Multi Detection System (Promega, Madison, WI, USA) with excitation and emission wavelengths of 530 nm and 575 nm, respectively. Triolein (Sigma-Aldrich, Saint Louis, MO, USA) was used to make a standard curve. The neutral lipid content (NL) was calculated according to the formula:
The lipid accumulation in transgenic strains were observed by a Nikon 80 i fluorescence microscope (Nikon, Kyoto, Japan) after being stained with Nile red. The excitation and emission wavelengths of 480 nm and 580 nm were applied, respectively.
For neutral lipid content (NL) examination, cells were stained by 0.5 µg/mL of Nile red (Sigma-Aldrich, Saint Louis, MO, USA) dissolved in 25% (v/v) of DMSO solution. The fluorescence intensity (FI) was measured with a Glomax-Multi Detection System (Promega, Madison, WI, USA) with excitation and emission wavelengths of 530 nm and 575 nm, respectively. Triolein (Sigma-Aldrich, Saint Louis, MO, USA) was used to make a standard curve. The neutral lipid content (NL) was calculated according to the formula:
The lipid accumulation in transgenic strains were observed by a Nikon 80 i fluorescence microscope (Nikon, Kyoto, Japan) after being stained with Nile red. The excitation and emission wavelengths of 480 nm and 580 nm were applied, respectively.
4
Quantitative Adipogenesis Assay Using 3T3-L1 Cells
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The 3T3-L1 cells were seeded into 96-well clear bottom black tissue culture treated plates (Corning) and differentiated as described above for 8 days with the indicated treatments. Medium was then removed and cells fixed by addition of 100 μl 4% paraformaldehyde (Thermo Fisher Scientific). Cells were then stained for intracellular lipid accumulation by adding 50 μl of 1 µg/ml Nile Red (Yorlab, York, UK) and 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotechnology) in 0.2% Triton X-100-phosphate-buffered saline for 15 min in the dark (Boucher et al., 2015 (link)). Nile Red staining for lipid droplets and DAPI staining for cell nuclei were imaged at 530 and 405 nm, respectively, using fluorescence imaging on a Nikon Eclipse Ts2 microscope (40× objective).
5
Quantitative Neutral Lipid Assay
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Neutral lipid (NL) was measured based on fluorescence intensities of 7-day-old cells staining with Nile Red (Sigma-Aldrich) according to a modified method reported by Chen et al. (2009) (link). Liquid cell cultures were mixed with Nile Red and DMSO with a final concentration of 5 μg/ml and 20% (v/v), respectively. After being incubated in darkness for 15 min, mixtures were directly measured by a Glomax-Multi Detection System (Promega, Madison, WI, United States), with excitation and emission wavelengths of 530 and 575 nm, respectively. The standard curve between the concentration of neutral lipid (μg/ml) and the Nile Red fluorescence level was made based on using Triolein (Sigma-Aldrich) as a reference standard. NL contents (μg/106 cells) were calculated using the following formula: NL(g/106cells) = cell0004×FI(530/575)−0.0038]×50/celldensity. For lipid drop observation, cells were cultured for 7 days and stained with Nile Red dye, then observed with a Nikon Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan) using an excitation and emission wavelength of 480 and 560 nm, respectively.
6
Visualizing Lipid Accumulation in 3T3-L1 Cells
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3T3‐L1 cells were seeded into 96‐well clear bottom black tissue culture‐treated plates (Corning) and differentiated as described above for 8 days with the indicated treatments. Medium was removed and cells were fixed by the addition of 100 μl of 4% paraformaldehyde (Thermo Fisher Scientific). Cells were then stained for intracellular lipid accumulation by adding 50 μl of 1 μg ml−1 Nile Red (Yorlab) and 1 μg ml−1 DAPI (Santa Cruz Biotechnology) in 0.2% Triton X‐100 phosphate‐buffered saline for 15 minutes in the dark (Boucher, Boudreau, Ahmed, & Atlas, 2015 ). Nile Red staining for lipid droplets and DAPI staining for cell nuclei were imaged at 530 and 405 nm respectively, using fluorescence imaging on a Nikon Eclipse Ts2 microscope (40× objective) (Kingston Upon Thames, UK).
7
Microscopic Observation of P. putida
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Example 7
Microscopic Observation of P. putida.
Microscopic observation of mcl-PHAs in P. putida by epifluorescence was performed by removing 1 mL from FPF-containing shake flask cultures after 48 hours. The cells were pelleted by centrifugation at 13,000 rpm for 1 minute, washed twice with 1× phosphate buffered saline (PBS), resuspended in 1 mL PBS containing 10 μg/mL Nile Red (Molecular probes, Invitrogen Cooperation, USA), and incubated at room temperature in the dark for 30 minutes. The cells were pelleted again, washed with 1×PBS, and resuspended in 1 mL PBS. 5 μL of resuspended cells were mixed with 5 μL of 1% (w/v) low-melting-temperature agarose to immobilize the cells, which were then placed on a microscopic slide with coverslip. Nile Red fluorescence was observed with band-pass filtering between 560-590 nm using a Nikon Eclipse 80i microscope (Nikon Corp., Japan).
8
Lipid Content Measurement in Oocytes
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DOs (5 replicates/group) were washed in PBS+PVA, fixed in 4% paraformaldehyde (PFD) and simultaneously permeabilized with 0.5% Triton X-100 solution in PBS+PVA for 15 min at room temperature and then washed again 3x in PBS+PVA. Oocytes were stained with 1 μg/mL Nile Red (N3013 – Molecular Probes, Life Technologies, Eugene, USA) in PBS+PVA for 30 min, protected from light and at room temperature. After this period, the oocytes were washed 3x in PBS+PVA and transferred to a glass slide (12 oocytes/slide) containing 5μL Pro Long Gold (Invitrogen), which was carefully covered with a coverslip. The fluorescence of Nile Red was evaluated under an epifluorescence microscope [Nikon Eclipse - TS100/filter G2A; excitation 515 to 560 nm and emission greater than 590 nm (Greenspan et al., 1985 (link)) with fast resolution (Romek et al., 2011 (link))]. All the photos obtained had their fluorescence intensities measured by the ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, MD). The photos were converted to gray scale (8 BIT) and the area of the oocyte to be analyzed was delimited. The lipid content in oocytes is presented as mean fluorescence intensity (FI) (Fu et al., 2011 (link)).
9
Confocal Imaging of Milk Components
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CLSM was carried out at each sampling time to monitoring microstructural changes of milk components. Milk samples were prepared according to [16] by staining with Fast Green FCF (Sigma-Aldrich, St Louis, USA) to observe the protein and Nile Red (Sigma-Aldrich) to observe the triacylglycerol core of fat globules. The stock solution of Fast Green was prepared at the concentration of 1 mg/mL in water (Millipore MilliQ), while the stock solution of Nile Red was prepared at the concentration of 1 mg/mL in water containing 0.8 mL/mL dimethyl sulfoxide (DMSO, Sigma-Aldrich). Both solutions were added 1:10 to milk at the time of staining. A sample volume of 8 µL was placed onto a microscopy slide and observed using an inverted confocal microscope (Nikon A1+ , 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, respectively. In dedicated sample preparations, the Hoechst 34580 was also added to milk at the final concentration of 12 µg/mL to stain bacteria cells. Hoechst 34580 was excited at a wavelength of 405 nm and the emission filter was set at 392-440 nm. Images were processed using the ImageJ software (NIH, USA).
10
Nile Red Staining for Lipid Droplet Quantification
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(1.5×105 cells/mL) were seeded on coverslips and cultured for 24 h. The cells were then washed twice with PBS and incubated with 5 μM Nile Red (Sigma, N3013) and 1 μg/mL Hoechst 33342 (Invitrogen, H1399) in RPMI 1640 complete medium for 30 min. THP-1 (1.5×105 cells/mL) were seeded in suspension and after 24 h they were pelleted, washed twice with PBS and incubated for 30 min in RPMI 1640 complete medium containing Nile Red and Hoechst at above indicated concentrations. For both cell lines, after staining, the medium was discarded and substituted with complete medium without phenol red. For imaging, THP-1 cells were placed on a microscope slide and "coverslipped". LDs and nuclei were acquired in live cells with a digital imaging system, using an inverted epifluorescence microscope with ×63/1.4 numerical aperture (NA) oil objective (Nikon Eclipse Ti-U, Nikon), applying 500 ms and 100 ms exposure time, for Nile Red and Hoechst, respectively. Images were captured with a back-illuminated Photometrics Cascade CCD camera system (Roper Scientific) and elaborated with Metamorph Acquisition/Analysis Software (Universal Imaging Corp). The number and the dimension of LDs were quantified using ImageJ software (51) . Statistical analysis comparing A2780 and THP-1 cells was performed by applying a two-tailed Student's T-test.
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