Here’s Why Neuroscience Researchers use TrueBlack® Lipofuscin Autofluorescence Quencher instead of Sudan Black B

Here’s Why Neuroscience Researchers use TrueBlack® Lipofuscin Autofluorescence Quencher instead of Sudan Black B

Here’s Why Neuroscience Researchers use TrueBlack® Lipofuscin Autofluorescence Quencher instead of Sudan Black B

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Neuroscience Brain

Background autofluorescence is a major issue for immunofluorescence imaging of cells and tissues, making imaging analysis complicated. TrueBlack® Lipofuscin Autofluorescence Quencher, an innovative reagent developed by Biotium can effectively quench lipofuscin autofluorescence in tissue sections and can also help to reduce autofluorescence from other sources like extracellular matrix.  

Sudan Black B has been traditionally used to quench lipofuscin autofluorescence. However, Sudan Black B also introduces non-specific background fluorescence in the red/far-red channels. TrueBlack® Lipofuscin Autofluorescence Quencher on the other hand not only masks the lipofuscin autofluorescence but also has a minimal increase in red/far-red background fluorescence. TrueBlack® Lipofuscin Autofluorescence Quencher treatment is compatible with conventional staining. It can be performed before or after immunostaining and has minimal effect on signal from fluorescent antibodies or nuclear counterstains, preserving the signal-to-noise ratio of the immunostaining.

TrueBlack® Lipofuscin Autofluorescence Quencher has been validated in several peer-reviewed publications and is widely used in several applications, especially in neuroscience research. Here are some reported studies using TrueBlack® Lipofuscin Autofluorescence Quencher in diverse neuronal cell types and tissues.

TrueBlack® Lipofuscin Autofluorescence Quencher Effectively Reduced Autofluorescence in Microglia

Zhang, H., Tan, C., Shi, X., & Xu, J. (2022). Impacts of autofluorescence on fluorescence-based techniques to study microglia. BMC Neuroscience, 23(1), 1-12. https://doi.org/10.1186/s12868-022-00703-1  

This photomicrograph of a brain tissue specimen, revealed the presence of a glial (microglial) nodule, consisting of numbers of microglial cells congregated about a blood vessel.

Microglia are a type of immune cell found in the central nervous system (CNS) that account for about 10-15% of cells within the brain. They remove damaged neurons and are important for maintaining the health of the CNS. 

In a recent study in BMC Neuroscience, Zhang et al. studied the impacts of autofluorescence in microglia. Microglia in aged animals have been reported to accumulate lipofuscin-like autofluorescent granules. Due to their location and wide emission spectrum, these autofluorescent granules can be misidentified as fluorescent signals from immunofluorescence staining. Using TrueBlack® Lipofuscin Autofluorescence Quencher, Zhang et al., reported a significant reduction of the autofluorescence background in microglia of fixed brain slices from 2-month-old Cx3cr1GFP mice. The quencher also does not interfere with a panel of antibodies targeting microglial proteins.

Imaging of autofluorescent granules in microglia on fixed brain slices of Cx3cr1GFP mouse. (Left) Z-projected image stacks of microglia with and without TrueBlack® Lipofuscin Autofluorescence Quencher treatment. (Right) Complied values of autofluorescence from the images. (n =17 cells, from 3 mice) ± SD, ***p<0.001

Credit: Zhang, et al. https://doi.org/10.1186/ s12868-022-00703-1

  • TrueBlack® Lipofuscin Autofluorescence Quencher Improves Signal-to-Noise in Resin Embedding of Brain Tissue

Ren, M., Tian, J., Sun, Q., Chen, S., Luo, T., Jia, X., Jiang, T., Luo, Q., Gong, H., & Li, X., (2021). Plastic embedding for precise imaging of large-scale biological tissues labeled with multiple fluorescent dyes and proteins. Biomed. Opt. Express 12, 6730-6745. https://doi.org/10.1364/BOE.435120

Resin embedding is a widely used technique to preserve structural information for the visualisation of fine structures in three dimensions. However, chemical treatments used in resin embedding are incompatible with fluorescent dyes and can also lead to quenching of fluorescence signals and poor signal-to-noise ratio.

In a recent publication in Biomedical Optics, Ren et al. developed a protocol that used TrueBlack® Lipofuscin Autofluorescence Quencher for improved resin embedding of large biological tissues. They demonstrated that by using 0.7% TrueBlack® Lipofuscin Autofluorescence Quencher in the glycol methacrylate embedding medium (GMA-T), it can effectively reduce up to 90% of the autofluorescence background in the tissue while retaining much more fluorescent signal than Sudan Black B. This new approach to resin embedding of organs provided a reliable technology for the study of complex brain structures.

Images of lipofuscin autofluorescence in methanol-fixed adult human brain tissue sections. (Top row) Untreated tissue. Lipofuscin autofluorescent appears in all channels. (Middle row) Sudan Black B treated. Lipofuscin autofluorescent masked but introduced high background in red/far-red channels. (Bottom row) TrueBlack® Lipofuscin Autofluorescence Quencher. Lipofuscin masked with minimal increase in the background. Credit: Biotium

TrueBlack® Lipofuscin Autofluorescence Quencher is an Excellent Quencher for mRNA Detection in Hybridization Chain Reaction in Human Neuronal Populations

May-Zhang, A. A., Benthal, J. T., & Southard-Smith, E. M. (2022). Hybridization Chain Reaction for mRNA Localization in Single Cells from Mouse and Human Cryosections. Current Protocols, 2(5), e439.  https://doi.org/10.1002/cpz1.439

The hybridization chain reaction (HCR), is an isothermal signal amplification method for nucleic acid detection. HCR-fluorescence in situ hybridization (HCR-FISH) enables researchers to visualise the expression of multiple RNA targets. After several iterations, the current third-generation HCR (V3HCR) now allows clear detection of mRNA to subcellular or even single-molecule resolution. However, the widespread lipofuscin autofluorescence found in neuronal cell populations can interfere with the HCR signal and complicate analysis.

In a recent Current Protocols publication, May-Zhang et al. tested a panel of lipofuscin quenching methods (cupric sulfate, bleaching in Dent’s fixative, detergent extraction, Murray’s clearing, treatment with 8% SDS, and standard blocking with Sudan Black B, and TrueBlack® Lipofuscin Autofluorescence Quencher) in HCR assays. They reported that the TrueBlack® Lipofuscin Autofluorescence Quencher is the most effective quencher for eliminating lipofuscin autofluorescence without causing V3HCR signal loss in frozen tissues from mouse and human.

Images of formaldehyde-fixed human cortex cryosections stained with CF®488A anti-NeuN antibody conjugate (green) and DAPI (blue). Treatment of TrueBlack® Lipofuscin Autofluorescence Quencher helps reduce autofluorescence from lipofuscin (right) when compared to untreated tissue (left). Credit Biotium

Why complicate your imaging analysis? Start removing background autofluorescence by using TrueBlack® Lipofuscin Autofluorescence Quencher to facilitate your fluorescence-based assays and make your interpretations easier! Download the publication list to discover more validated applications for TrueBlack® Lipofuscin Autofluorescence Quencher.Learn more about TrueBlack® background reducers.


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  • May-Zhang, A. A., Benthal, J. T., & Southard-Smith, E. M. (2022). Hybridization Chain Reaction for mRNA Localization in Single Cells from Mouse and Human Cryosections. Current Protocols, 2(5), e439.  https://doi.org/10.1002/cpz1.439

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