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TrueBlack Lipofuscin Autofluorescence Quencher, 20X in DMF

TrueBlack™ is a new reagent for quenching lipofuscin autofluorescence in tissue sections for immunofluorescence staining. It can also help reduce autofluorescence from other sources like extracellular matrix.

TrueBlack Lipofuscin Autofluorescence Quencher, 20X in DMF
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23007

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1 mL

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Product Description

TrueBlack™ is a new reagent for quenching lipofuscin autofluorescence in tissue sections for immunofluorescence staining. Lipofuscin consists of highly autofluorescent granules of oxidized proteins and lipids that build up in the lysosomes of aging cells (1). Lipofuscin granules fluoresce brightly in all channels used for fluorescence microscopy, and accumulate in a variety of cell and tissue types with age. Consequently, imaging of specific immunofluorescence signal in certain adult human tissues or aged animal tissues can be virtually impossible unless methods are employed to quench or mask lipofuscin fluorescence.

Traditionally, Sudan Black B has been used to quench lipofuscin autofluorescence by incubating tissue sections with the dye after immunofluorescence staining (2). However, while it masks the autofluorescence from lipofuscin, Sudan Black B also introduces uniform non-specific background fluorescence in the red and far-red channels (Ref. 3 and Fig. 1 below), limiting the use of fluorescent dyes in those wavelengths. Now Biotium has developed TrueBlack™ as a superior alternative to Sudan Black B. TrueBlack quenches lipofuscin fluorescence with far less increase in red/far-red background fluorescence (Fig. 1). TrueBlack™ treatment can be performed before or after immunostaining. It is rapid, simple, and has minimal effect on signal from fluorescent antibodies or nuclear counterstains, thus preserving the signal-to-noise ratio of the immunostaining (Figs. 2-3).

TrueBlack effectively eliminates lipofuscin autofluorescence in tissues like human brain (Ref. 4 and Figs. 1-3 below) and human retina (Ref. 5).  TrueBlack also can reduce autofluorescence from other sources, such as collagen, elastin, red blood cells, and general background fluorescence. It is not as effective at quenching these sources of autofluorescence as it is for lipofuscin, but it can improve background in human and non-human tissue types (Figs. 4-5).

Also see our other accessory products for immunofluorescence staining and other applications.

Features:

  • Eliminates lipofuscin autofluorescence
  • Can reduce autofluorescence from non-lipofuscin sources
  • Doesn’t cause high background like Sudan Black B
  • Clears the way for fluorescence imaging of human and aged animal tissues

 

 TrueBlack™ quenches lipofuscin with less background than Sudan Black B

TrueBlack composite 2
Figure 1. Lipofuscin autofluorescence in methanol-fixed adult human tissue sections. In untreated tissue (top row), lipofuscin appeared as fluorescent granules that fluoresced in all fluorescence channels. Sudan Black B (middle row) masked lipofuscin autofluorescence, but introduced background in the red and far-red channels. TrueBlack (bottom row) masked lipofuscin with minimal increase in fluorescence background (bottom row). Methanol-fixed cryosections of adult human cerebral cortex were left untreated or stained with 0.1% Sudan Black B in 70% ethanol or 1X TrueBlack according to product protocol. Samples were imaged at the same gain settings on a Zeiss LSM 700 confocal microscope in the FITC (green), Cy3 (red), and Cy5 (far-red) channels.

 

TrueBlack™ treatment can be performed before or after immunofluorescence staining

Quenching of lipofuscin autofluorescence using TrueBlack pretreatment. Formaldehyde-fixed human cortex cryosections were left untreated (A) or treated with TrueBlack (B), then stained with CF488A anti-NeuN antibody conjugate (green) and DAPI (blue). Sections were imaged in all channels on a Zeiss LSM700 confocal microscope. Lipofuscin fluoresces brightly in all channels, appearing as white spots in the merged image of untreated tissue (A). TrueBlack pretreatment eliminated lipofuscin autofluorescence (B), with negligible effect on specific staining.
Figure 2. Quenching of lipofuscin autofluorescence using TrueBlack pretreatment before immunofluorescence staining. Formaldehyde-fixed human cortex cryosections were left untreated (A) or treated with TrueBlack (B), then stained with CF488A anti-NeuN antibody conjugate (green) and DAPI (blue). Sections were imaged in all channels on a Zeiss LSM700 confocal microscope. A. Lipofuscin fluoresces brightly in all channels, appearing as white spots (red arrows)  in the merged image of untreated tissue. B. TrueBlack pretreatment eliminated lipofuscin autofluorescence, with negligible effect on specific staining.

 

Trueblack CF640R GAR DAPI ihc composite
Figure 3. Quenching of lipofuscin autofluorescence using TrueBlack post-treatment after immunofluorescence staining. Methanol-fixed cryosections of human cerebral cortex were stained with rabbit anti-GFAP followed by CF640R goat anti-rabbit (cat. no. 20176) and DAPI (cat. no. 40009). Sections were left untreated or treated with TrueBlack, then mounted in EverBrite mounting medium (cat. no. 23001). Untreated sections (left) showed lipofuscin autofluorescence (white arrows) in addition to GFAP immunofluorescence (glial processes, magenta) and DAPI staining (nuclei, blue). TrueBlack (right) eliminated lipofuscin fluorescence while maintaining good signal-to-noise for GFAP immunostaining in the far-red channel (magenta) and nuclear counterstaining (blue).

 

TrueBlack™ can reduce autofluorescence from sources other than lipofuscin

TrueBlack reduces autofluorescence in human kidney sections in the FITC, Cy3, and Cy5 channels. Human kidney cryosections were fixed in ice-cold methanol and nuclei were stained with DAPI (blue). Autofluorescence was imaged on a Zeiss LSM700 confocal microscope in all channels with the same imaging settings for both samples.Top panel: untreated; Bottom panel: TrueBlack-treated.
Figure 4. TrueBlack reduces non-lipofuscin autofluorescence in human kidney sections in the FITC, Cy3, and Cy5 channels. Human kidney cryosections were fixed in ice-cold methanol and nuclei were stained with DAPI (blue). Autofluorescence was imaged on a Zeiss LSM700 confocal microscope in all channels with the same imaging settings for both samples.Top panel: untreated; Bottom panel: TrueBlack-treated.

 

TrueBlack reduces autofluorescence in the FITC channel in rat kidney tissue sections. Rat kidney cryosections were fixed in formaldehyde, and left untreated (A) or treated with TrueBlack (B). Nuclei were stained with DAPI (blue). Autofluorescence in the FITC channel (green) was imaged on a Zeiss LSM700 confocal microscope using the same imaging settings for each sample.
Figure 5. TrueBlack reduces non-lipofuscin autofluorescence in the FITC channel in rat kidney tissue sections. Rat kidney cryosections were fixed in formaldehyde, and left untreated (A) or treated with TrueBlack (B). Nuclei were stained with DAPI (blue). Autofluorescence in the FITC channel (green) was imaged on a Zeiss LSM700 confocal microscope using the same imaging settings for each sample.

References

1. Hohn, A. and Grune, T. Redox Biol 1(1): 140, 2013.

2. Schnell, S.A., Staines, W.A., and Wessendorf, M.W. J Histochem Cytochem 47(6): 719, 1999.

3. Romijn, H.J., van Uum, J.F.M., Breedijk, I., Emmering, J., Radu, I., and Pool, C.W. J Histochem Cytochem 47(2): 229, 1999.

4. Fosso M.Y., McCarty K., Head E., Garneau-Tsodikova S., and LeVine H. 3rd. ACS Chem Neurosci doi: 10.1021/acschemneuro.5b00266

5. Chan T., Zhu L., Madigan M.C., Wang K., Shen W., Gillies M.C., Zhou F. Br J Pharmacol.172(9): 2343, 2015.

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Support & faq

Product shelf life, solubility, shipping and storage, and stability

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

 

Category: GelRed™ and GelGreen™ Nucleic Acid Gel Stains, Mix-n-Stain™ Antibody Labeling Kits, DNA Quantitation Kits, NucView™ Caspase 3 Enzyme Substrates, Apoptosis, Necrosis, and Cell Viability Kits, Luciferase Assays, CF™ Dyes, Product shelf life, solubility, shipping and storage, and stability

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Many of our solid compounds are packaged by lyophilization, in which case they usually do not appear as fluffy powders, but form a film or coating on the sides of the vial. Simply add the appropriate volume of the recommended solvent to the vial to make the desired concentration stock solution, and swirl or gently vortex to mix. Make sure the solvent comes in contact with the inside walls of the vial to fully recover the product.

Category: Product shelf life, solubility, shipping and storage, and stability

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Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF™ dye succinimidyl ester stability
Succinimidyl esters are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® are particularly hygroscopic, worsening the hydrolysis problem. For example, the percent of active Alexa Fluor® 488 succinimidyl ester (SE) could be well below 50% by the time of application (according to the manufacturer’s product datasheet). In a number of Alexa Fluor® SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while in all of Biotium’s CF™ dyes the SE group is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF™ dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Category: Mix-n-Stain™ Antibody Labeling Kits, DNA Quantitation Kits, NucView™ Caspase 3 Enzyme Substrates, Luciferase Assays, CF™ Dyes, Product shelf life, solubility, shipping and storage, and stability

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Shipping/Shelf-life

Shipment Method: Shipping and handling methods will be assessed and calculated at time of shipment based upon item(s) storage temperature conditions. Expedited shipment may be requested at time of checkout. Please note that products with recommended storage at 4°C or -20°C may ship at ambient temperature. This will not affect product performance. When you receive the product, place it under the recommended storage conditions.