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TrueBlack Lipofuscin Autofluorescence Quencher

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)

Features:

  • Eliminates lipofuscin autofluorescence
  • Can reduce autofluorescence from non-lipofuscin sources
  • Has low red/far-red background compared to Sudan Black B
  • Can be used before or after immunostaining
  • Clears the way for fluorescence imaging of human and aged animal tissues

Ordering information:

Catalog numberProductUnit sizeNotes
23007 TrueBlack Lipofuscin Autofluorescence Quencher, 20X in DMF 1 mL1 mL treats ~200 tissue sections

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

 

TrueBlack™ quenches lipofuscin with less background than Sudan Black B

TrueBlack composite 2
Figure 1. Lipofuscin autofluorescence in methanol-fixed adult human brain 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 brain cerebral 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.