Support

Quick links
Frequently asked questions - categories
Ask a question
Contact Us
+1

TrueBlack®

Clears the Way for Immunofluorescence in Human Tissue

Eliminates lipofuscin autofluorescence
Low red background compared to Sudan Black B
Clears the way for fluorescence imaging

Lipofuscin can make fluorescence staining of human tissues virtually impossible
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. 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.

TrueBlack® treatment is simple and rapid
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.

Unlike Sudan Black, TrueBlack® quenches autofluorescence with minimal background
Traditionally, Sudan Black B has been used to quench lipofuscin autofluorescence by incubating tissue sections with the dye after immunofluorescence staining. 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, limiting the use of fluorescent dyes in those wavelengths. Biotium developed TrueBlack® as a superior alternative to Sudan Black B to quench autofluorescence with far less increase in red/far-red background fluorescence.

TrueBlack® helps reduce autofluorescence from other sources too
TrueBlack® effectively eliminates lipofuscin autofluorescence in tissues like human brain and retina. 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. It also has been used to quench background fluorescence on polycarbonate filters used as cell supports for imaging cytometry (Futia et al. 2016).

VIEW & PURCHASE

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, but introduced high background in the red and far-red channels. TrueBlack® (bottom row) masked lipofuscin with minimal increase in background.

TrueBlack® treatment can be performed before or after antibody 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. TrueBlack® pretreatment before immunofluorescence staining. Formaldehyde-fixed human cerebral cortex cryosections were left untreated (A) or treated with TrueBlack® (B), then stained with CF488A anti-NeuN antibody conjugate (green) and DAPI (blue). 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® 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 3. TrueBlack® reduces non-lipofuscin autofluorescence in human kidney sections in the FITC, Cy®3, and Cy®5 channels. Human kidney cryosections were fixed in ice-cold methanol and nuclei were stained with DAPI (blue).Top panel: untreated; Bottom panel: TrueBlack®-treated.

TrueBlack® also can improve background in non-human tissue

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 4. 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.

Cy3 and Cy5 are registered trademarks of GE Healthcare.

TrueBlack Lipofuscin Autofluorescence Quencher

From: $110 Sizes: 1 mLCatalog #: 23007