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PC6S and DBC30 are low-molecular-weight compounds that exhibit strong fluorescence when dissolved in lipid droplets. They can be used not only with cultured cells but also with tissues to clearly and distinctly stain lipid droplets. Because of their excellent droplet selectivity and prolonged intracellular retention, they can be used alongside multiple other fluorescent probes. PC6S emits green fluorescence (λex, max = 468 nm, λem, max = 535 nm, in dibutyl ether) and can be visualized with GFP or FITC filter sets. DBC30 emits blue fluorescence (λex, max = 403 nm, λem, max = 468 nm in dibutyl ether) and can be visualized with DAPI filter sets.
Products
‡ These products were developed by Dr. Toshitada Yoshihara.
Excitation and Emission Spectra
λex max = 468 nm, λem max = 535 nm (in dibutyl ether)
Compatible with optical filter for DAPI.
λex max = 403 nm, λem max = 468 nm (in dibutyl ether)
Compatible with optical filter for GFP or FITC.
Application: multiple staining of cells
Figure. A : PC6S (Lipid droplet), B : Hoechst® 33342 (Nucleus), C : MitoTracker® Red (Mitochondria), D : Merged image, Each scale bar is 20 μm.
‡These figures are provided by Dr. Toshitada Yoshihara.
Procedure
- Add oleic acid to medium of HeLa cells and incubate for 24 hours.
- Replace with medium containing PC6S, Hoechst® 33342, MitoTracker® Red and incubate at 37 °C for 30 minutes.
- Remove the medium and wash twice with PBS(-).
- Observe through fluorescence microscope.
Hoechst® is a registered trademark of Hoechst GmbH. MitoTracker® is a registered trademark of Molecular Probes, Inc.
Application: staining of lipid droplets by PC6S and DBC30 in vivo
Figure. Mouse Liver Surface Images
A: Healthy Mouse Liver, B: Liver from Fatty-Liver Model Mouse, Blue: DBC30, Green: PC6S
All figures were provided by Dr. Toshitada Yoshihara.
Procedure
- Preparation of Fatty-Liver Model Mice
- Feed mice a choline-deficient, methionine-restricted, ultra-high-fat diet for one week.
- Acquisition of Lipid-Droplet Images with DBC30 or PC6S
- While the mice are under anesthesia, inject 50 nmol of DBC30 or PC6S into the tail vein.
- After 30 minutes, perform a laparotomy and observe the liver with a confocal fluorescence microscope.
DBC30: excitation 405 nm; emission 440 – 480 nm
PC6S: excitation 488 nm; emission 500 – 540 nm
Reference
- Visualization of Lipid Droplets in Living Cells and Fatty Livers of Mice Based on the Fluorescence of π-Extended Coumarin Using Fluorescence Lifetime Imaging Microscopy
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Example of staining with Oil Red O
Oil red O is a diazo dye which has been widely used for staining fat cell and lipids since long ago. This lysochrome (fat-soluble dye) red dye can be used for staining of neutral triglycerides and low polar lipids. Oil Red O staining is done on fresh or frozen samples, since alcohol fixation removes lipid. Its usage is simple, and requires only washing after adding the staining solution, which makes it easy to identify lipids visually.
Furthermore, accumulation of lipids can be quan-tified by eluting the dye using isopropanol after staining and measuring the absorbance.
Figure. 10 days later of addition of media for differentiation to 3T3-L1 cell and then stained by 1 mg/mL of O0483.