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TRUEBLACK® BACKGROUND REDUCERS

Block fluorescence background from multiple sources for IF and WB

Non-specific fluorescence background can have several causes, including sample autofluorescence, non-specific antibody binding, and electrostatic interactions of fluorescent dyes with proteins and blotting membranes. TrueBlack® reagents for IF staining and western blotting block background from multiple sources for optimal specificity.

Clears the way for immunofluorescence of human tissue
Blocks IF background from multiple sources
Superior blocking for fluorescent/near-IR westerns

TrueBlack® Lipofuscin Autofluorescence Quenchers

Lipofuscin can make fluorescence imaging of human tissues virtually impossible

Lipofuscin consists of highly autofluorescent granules of oxidized proteins and lipids that build up in the lysosomes of aging cells in a variety of tissues. Lipofuscin granules fluoresce brightly in all channels used for fluorescence microscopy (Figure 1). Consequently, immunofluorescence in many human tissues or aged animal tissues can be virtually impossible unless lipofuscin fluorescence is masked.

TrueBlack® eliminates lipofuscin autofluorescence, clearing the way for immunofluorescence

Traditionally, Sudan Black B has been used to quench lipofuscin autofluorescence by incubating tissue sections with the dye after immunofluorescence staining. However, Sudan Black B also introduces non-specific red and far-red fluorescence,  limiting the use of fluorescent dyes in those wavelengths. Biotium developed TrueBlack® Lipofuscin Autofluorescence Quencher as a superior alternative to Sudan Black B to quench autofluorescence with much lower background.

TrueBlack®  Lipofuscin Autofluorescence Quencher Features

  • Eliminates lipofuscin autofluorescence
  • Reduces autofluorescence from non-lipofuscin sources
  • Doesn’t cause high background, unlike Sudan Black B
  • Can be used before or after immunofluorescence staining
  • Clears the way for fluorescence imaging of human and aged animal tissues

TrueBlack® treatment can be performed before or after immunostaining (Figures 2-3). It is rapid, simple, and has minimal effect on signal from fluorescent antibodies or nuclear dyes, thus preserving specific staining. Quenching is stable and compatible with commonly used wet-set and hardset fluorescence mounting media, so slides can be stored after staining. See TrueBlack® FAQs for more information.

TrueBlack® reduces 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, and red blood cells (Figures 4-5). 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 fluorescence on polycarbonate filters used as cell supports for imaging (Futia et al. 2016). Download a list of TrueBlack® references.

 

TrueBlack® eliminates lipofuscin fluorescence 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® autofluorescence quencher can be used before or after antibody staining

TrueBlack Lipofuscin Autofluorescence Quencher pre-treatment
Figure 2. TrueBlack® treatment before staining preserves antibody brightness. Human cerebral cortex was left untreated or treated with TrueBlack®, then stained with CF®488A anti-NeuN (neuronal nuclei, green) and DAPI (nuclei, blue). In untreated tissue, lipofuscin granules appeared as white speckles (left). TrueBlack® pre-treatment quenched lipofuscin fluorescence without affecting specific staining (right).
TrueBlack Lipofuscin Autofluorescence Quencher post-treatment
Figure 3. TrueBlack® treatment can be performed after immunofluorescence staining. Human brain sections were stained with rabbit anti-GFAP antibody and CF®640R goat anti-rabbit (glia, magenta) and DAPI (nuclei, blue). The sections were imaged without further treatment (left) or after TrueBlack® treatment (right). Autofluorescent lipofuscin granules in untreated tissue are marked with arrows.


TrueBlack® quencher can reduce autofluorescence from multiple sources

TrueBlack Autofluorescence Quencher human kidney
Figure 4. TrueBlack® can reduce non-lipofuscin autofluorescence. Untreated human kidney sections showed autofluorescence from multiple sources including extracellular matrix and blood cells (top), which was reduced by TrueBlack® treatment (bottom).
TrueBlack Autofluorescence Quencher rat kidney
Figure 5. TrueBlack® can reduce autofluorescence in non-human tissue. Rat kidney sections were left untreated or treated with TrueBlack®, then stained with DAPI (nuclei, blue). Untreated tissue showed green autofluorescence from extracellular matrix (left), which was quenched by TrueBlack® (right).

TrueBlack® is widely published for reducing lipofuscin autofluorescence in several human and animal tissues.

Highlighted Citation: In a publication in Biological Procedures Online, Axelrod et al. applied TrueBlack® Lipofuscin Autofluorescence Quencher to enhance imaging of circulating tumor cells (CTCs) in blood and disseminated tumor cells (DTCs) in bone marrow, allowing multiplex target detection and cell morphology analysis of extremely rare cells.

See the full list of references here.

Effect of TrueBlack® on background signal due to autofluorescence. Samples were stained with pan cytokeratin and white blood cell markers. Autofluorescent signal reduction is most evident in the AF555 channel (where no primary or secondary antibody was applied) and in the AF488 channel (stained for pan cytokeratin). Filled white arrowheads point to true positive staining. Scale bar = 50 μm. Credit: Axelrod et. al. doi:10.1186/s12575-018-0078-5.


TrueBlack® Plus: A unique lipofuscin quencher

TrueBlack® Plus is a next-generation lipofuscin quencher developed by Biotium chemists. This new quencher was designed to allow lipofuscin quenching in aqueous buffer with even lower background than the original TrueBlack®. Quenching in PBS allows longer incubation times for thick samples without shrinkage, and is compatible with hydrophobic stains.

TrueBlack® Plus Lipofuscin Autofluorescence Quencher Features

  • Quenches lipofuscin with lower far-red background than our original TrueBlack®
  • The only lipofuscin quencher that can be used in aqueous buffer instead of 70% EtOH
  • Reduces autofluorescence from non-lipofuscin sources
  • Lower background than traditional Sudan Black B treatment
  • Fast and simple treatment before or after immunostaining
  • Stable quenching, compatible with commonly used fluorescence mounting media
  • Clears the way for multi-color imaging in human tissue

TrueBlack® Plus Quenches lipofuscin with lower far-red background than our original TrueBlack®

Figure 6. Immunofluorescence staining in human cerebral brain with or without lipofuscin quenching. Methanol-fixed cerebral cortex cryosections were stained with CF®405S GFAP conjugate (clone ASTRO/1974R), then left untreated, or treated with original TrueBlack® Lipofuscin Autofluorescence Quencher in 70% EtOH, or TrueBlack® Plus Quencher in PBS. GFAP fluorescence appears as blue filamentous staining, while lipofuscin appears as bright punctate spots (pink/orange in the merged images). TrueBlack® Plus selectively quenches lipofuscin autofluorescence, with minimal far-red background. Top: merge of all channels showing GFAP signal and lipofuscin autofluorescence. Bottom: merge of green, red, and far-red channels showing background fluorescence.


Choose the right quencher for your application

Comparison of original TrueBlack® Lipofuscin Autofluorescence Quencher and TrueBlack® Plus

ProductCatalog no.Supplied asProsCons
TrueBlack® Lipofuscin Autofluorescence Quencher2300720X in DMF• Complete quenching of lipofuscin autofluorescence

• Ultra-low background in blue and green channels

• Quenching takes only 30 seconds
• Introduces some red/far-red background

• Quenching must be done in 70% EtOH

• Some quenching of fluorescent dyes
TrueBlack® Plus Lipofuscin Autofluorescence Quencher2301440X in DMSO• Greatly reduces lipofuscin autofluorescence

• Has lower red/far-red background than the original TrueBlack®

• The only lipofuscin quencher that can be used in PBS and other aqueous buffers
• Titration recommended for optimal quenching

• May not be as effective as the original TrueBlack® for high-lipofuscin samples

• Some quenching of fluorescent dyes

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TrueBlack® IF Background Suppressor System

The TrueBlack® Background Suppressor System is a buffer system designed for optimal blocking of non-specific staining for immunofluorescence (IF). The buffers are designed to block background from both non-specific antibody binding as well as direct interaction of fluorescent dyes on antibodies with cells or tissue sections.

TrueBlack® IF Background Suppressor Features

  • Suppresses background from non-specific antibody binding and charged fluorescent dyes
  • More efficient than Image-iT® FX, block & permeabilize in just 10 minutes
  • Complete system for blocking, permeabilizing, and antibody dilution
  • Non-mammalian blocking agents, for broad secondary antibody compatibility
  • For immunofluorescence on cells or tissue sections

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TrueBlack® IF Background Suppressor System (Permeabilizing) – 20 assays
TrueBlack® IF Background Suppressor System (Permeabilizing) – 200 assays

 

Charged dyes can contribute to antibody background

TrueBlack IF Background Suppressor
Figure 1. TrueBlack® IF Background Suppressor reduces non-specific binding of antibodies conjugated to charged fluorescent dyes. Methanol-fixed HeLa cells were blocked for 10 minutes with fish gelatin blocking buffer or TrueBlack® IF Background Suppressor, then stained with no primary antibody or mouse anti-tubulin antibody, followed by goat anti-mouse secondary antibody conjugated to the indicated dyes. Antibodies were diluted in the same buffer used for blocking.


Fluorescent dyes can be an unexpected cause of non-specific antibody binding

Non-specific signal in immunofluorescence can arise from multiple sources, including antibody cross-reactivity with off-target proteins, non-specific antibody adsorption to the sample, and tissue autofluorescence. Another cause of background that is not widely known is the effect of fluorescent dyes themselves on the antibody specificity. Next-generation fluorescent dyes like Alexa Fluor® or CF® dyes often carry multiple negative charges to improve dye solubility and brightness of conjugates. However, the extra charge carried by the dye can result in non-specific antibody binding that can reduce the signal-to-noise ratio of immunostaining, particularly for low abundance targets. While conventional blocking agents like BSA or gelatin can reduce non-specific protein binding, they don’t block background from charged dyes (Figure 1).

TrueBlack® Background Suppressing System blocks multiple sources of antibody background

TrueBlack® Background Suppressor includes reagents for blocking both non-specific protein binding as well as background from charged dyes. Examples of charged dyes that show improved signal to noise with the Background Suppressor are CF®405S, CF®405M, CF®555, Alexa Fluor® 647, and Cy®5.5 (Figures 1-3). One-step blocking and permeabilization takes only 10 minutes, and the buffers contain no mammalian proteins, for broad antibody compatibility.

Excellent blocking for Mix-n-Stain™ labeled antibodies

TrueBlack IF Background Suppressing System Background Suppressor CF555 Mi-n-Stain
Figure 3. TrueBlack® IF Background Suppressor blocks non-specific background from the charged dye CF®555. Methanol-fixed HeLa cells were blocked for 10 minutes with fish gelatin blocking buffer or TrueBlack® IF Background Suppressor, then stained with CF®555 Mix-n-Stain™-labeled anti-nucleolin antibody clone NCL/902 diluted in the same buffer used for blocking. The CF®555 labeled antibody showed non-specific cytoplasmic staining with gelatin blocking buffer, but Background Suppressor restored specific nucleolar staining.

TrueBlack® Background Suppressor is as effective as Image-iT® FX

TrueBlack Background Suppressing System Background Suppresor vs Image-iT FX Signal Enhancer
Figure 2. TrueBlack® IF Background Suppressor blocks non-specific binding from charged dyes like Alexa Fluor® 647 as effectively as Image-iT® FX. Methanol-fixed HeLa cells were blocked with the indicated blocking buffer, then incubated with Alexa Fluor® 647 goat anti-mouse secondary diluted in the same blocking buffer (gelatin blocking buffer was used for the Image-iT® FX sample).

For immunofluorescence blocking of cells or tissue sections

CF555 Mix-n-Stain NucSpot 470 TrueBlack Background Suppressing System Background Suppressor intestine
Figure 4. Rat intestine cryosection blocked with TrueBlack® IF Background Suppressor (Permeabilizing) and stained with CF®555 Mix-n-Stain™ labeled anti-ZO1 (tight junctions, red) and NucSpot® 470 nuclear stain (nuclei, green), diluted in TrueBlack® IF Blocking Buffer (Permeabilizing) for staining.

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Alexa Fluor and Image-iT are registered trademarks of Thermo Fisher Scientific. Cy Dye is a registered trademark of GE Healthcare.

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TrueBlack® WB Blocking Buffer Kit

The TrueBlack® WB Blocking Buffer Kit is a ready-to-use buffer system for fluorescence-based western blotting (WB). The buffers yield optimal specificity and sensitivity by blocking non-specific interactions of dye-labeled antibodies with proteins and the blotting membrane.

TrueBlack® WB Blocking Buffer Kit Features

  • Blocks as well or better than Odyssey® Blocking Buffer, at a lower price
  • Reduces non-specific protein bands and background over entire membrane
  • Suppresses background from charged dyes better than BSA, gelatin, or casein
  • Compatible with PVDF and nitrocellulose membranes
  • Contains no mammalian proteins, for broad antibody compatibility
  • For visible and near-IR fluorescent westerns
Figure 1. Western detection of phospho-Erk1/2 in PDGF-stimulated NIH-3T3 cell lysate. Membranes were blocked with fish gelatin blocking buffer, LI-COR® Odyssey® TBS Blocking Buffer, or TrueBlack® WB Blocking Buffer. Phosphorylated Erk was detected using rabbit anti-pErk1/2 and CF®680R donkey anti-rabbit antibodies. TrueBlack® WB Blocking Buffer gave lower background fluorescence and better specificity compared to the other buffers.


Superior western blocking for next-generation fluorescent dyes

Non-specific signal in WB can arise from multiple sources, including antibody cross-reactivity with off-target proteins, non-specific antibody adsorption to the membrane, and membrane autofluorescence. Another potential cause of background is the effect of fluorescent dyes themselves on the specificity of labeled antibodies. Highly charged dyes like Alexa Fluor® or CF® dyes have improved solubility and brightness of conjugates compared to uncharged dyes. However, the extra charge carried by antibodies labeled with these dyes can result in non-specific binding to proteins and membranes. The TrueBlack® WB Blocking Buffer Kit blocks background from multiple sources including charged dye conjugates (Figure 2). TrueBlack® blocking buffer is especially advantageous for phosphoprotein detection, significantly improving specificity compared to conventional blocking buffers (Figure 1).

Switch from Odyssey® Blocking Buffer and save

TrueBlack® WB Blocking Buffer performs as well or better for fluorescent WB compared to LI-COR’s Odyssey® Blocking Buffer (Figure 1), and is priced lower on a per membrane basis.

Compare TrueBlack® WB Blocking Buffer Kit with Odyssey® Blocking Buffer

ProductTrueBlack® WB Blocking
Buffer Kit
Odyssey® Blocking Buffer
Trial SizeFor 10 membranes125 mL for 4 membranes
Full SizeFor 50 membranes500 mL for 16 membranes
Number of membranes if Odyssey® Blocking Buffer is used for each blocking and antibody dilution step. TrueBlack® WB Blocking Buffer Kit includes enough buffers for all blocking and antibody incubation steps for the stated number of membranes.
Figure 2. Western detection of tubulin in HeLa cell lysate with mouse anti-tubulin and Alexa Fluor® 790 goat anti-mouse antibodies. Membranes were blocked with fish gelatin blocking buffer or TrueBlack® WB Blocking Buffer. The highly negatively charged Alexa Fluor® 790 labeled antibody showed non-specific binding to the PVDF membrane and cellular proteins, which was blocked by TrueBlack® WB Blocking Buffer. Lanes 1-3: 10 ug, 1 ug, or 0.1 ug HeLa cell total protein. Note: Alexa Fluor ®790 dye was used to demonstrate non-specific background from highly charged dyes. Biotium’s near-IR CF® dyes are peggylated, a structural feature that renders the dyes highly water soluble without excessive negative charge, resulting in significantly lower background. Learn more about Near-IR CF® Dyes.

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TrueBlack® WB Blocking Buffer Kit – 10 membranes
TrueBlack® WB Blocking Buffer Kit – 50 membranes

LI-COR and Odyssey are registered trademarks of LI-COR Inc.

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