TrueBlack® Background Reducers
TrueBlack® Lipofuscin Quencher can be used with the TrueBlack® IF Background Suppressor (Permeabilizing) kit in an immunofluorescence staining workflow, to reduce background from both non-specific antibody and dye binding, and autofluorescence arising from the sample itself. Because TrueBlack® IF reagents contain detergent, the TrueBlack® lipofuscin quencher would need to be used after immunostaining (Post-treatment protocol) as the latter is not compatible with detergents present in the IF reagents.
If immunostaining is performed with buffers lacking detergents, the TrueBlack lipofuscin quencher may be used before (Pre-treatment protocol) or after immunostaining.
TrueBlack® Lipofuscin Autofluorescence Quencher and TrueBlack® Plus Lipofuscin Autofluorescence Quencher were not designed for colorimetric detection, but they will stain lipofuscin black and be visible in light microscopy.
The mechanism of action of TrueBlack® and TrueBlack® Plus Lipofuscin Autofluorescence Quenchers is similar to the traditionally used dye Sudan Black B. Lipofuscin consists of autofluorescent granules of oxidized proteins and lipids that build up in the lysosomes of cells as a consequence of aging. The TrueBlack® lipofuscin quenchers are hydrophobic in nature and associate with the lipid rich lipofuscin to quench autofluorescence.
TrueBlack® and TrueBlack® Plus Lipofuscin Quenchers have similar properties as Sudan Black B and, therefore, primarily stain lipofuscin. Like Sudan Black B, they may also stain some myeloid cells, as well as lipid droplets, but we have not confirmed this.
TrueBlack® Plus Lipofuscin Autofluorescence Quencher is the only lipofuscin quencher that can be used in aqueous buffer instead of 70% EtOH, allowing longer incubation times for thick tissue samples without shrinkage. TrueBlack® Plus offers even lower far-red background than the original TrueBlack® Lipofuscin Autofluorescence Quencher. It is recommended to review the staining protocol of TrueBlack® and TrueBlack® Plus to decide between the two.
While customers have reported success using TrueBlack® Lipofuscin Autofluorescence Quencher to quench RBC autofluorescence, during in-house research we’ve only ever seen partial quenching for RBCs. Experimental samples can vary quite a lot in the intensity of autofluorescence depending on age and processing, so the effectiveness of RBC quenching may be variable.
When we test TrueBlack® Lipofuscin Autofluorescence Quencher in an assay, we evaluate lipofuscin quenching in human brain sections, but our standard QC for the dye is measuring purity and OD of the solution, not tissue staining.
Alternative products that may be more effective at quenching RBCs or ECM would be TrueBlack® Plus (Cat. No. 23014) or EverBrite™ Hardset with TrueBlack® (Cat. No. 23017-23019) however, these products have not been empirically evaluated for this application.
TrueBlack® Lipofuscin Autofluorescence Quencher (Cat. No. 23007) and TrueBlack® Plus Lipofuscin Autofluorescence Quencher (Cat. No. 23014) are designed to reduce autofluorescence from lipofuscin in tissue samples such as mouse and human brains and retina. While TrueBlack® Lipofuscin Quenchers have been reported to reduce autofluorescence from other sources, such as collagen, elastin, red blood cells, and general background fluorescence, they are not as effective at quenching these types of autofluorescence as for lipofuscin autofluorescence. However, they may improve background from a variety of sources in different experimental systems.
TrueBlack® IF Background Suppressor System (Cat. No. 230120) is a buffer system designed for optimal blocking of non-specific antibody binding as well as direct interaction of fluorescent dyes on antibodies with cells or tissue sections to eliminate non-specific staining for immunofluorescence (IF).
The TrueBlack® WB Blocking Buffer Kit (Cat. No. 23013) is a ready-to-use buffer system for blocking non-specific interactions of dye-labeled antibodies with proteins and the blotting membrane in fluorescence-based western blotting (WB).
Dyes that carry multiple negative charges can introduce background. Usually, this is more of a concern with labeled antibodies that carry many dyes, as opposed to a small toxin like bungarotoxin. When staining tissues, the endogenous autofluorescence of the tissue itself is often the most significant source of background. Endogenous fluorescence background in tissue is usually highest in the blue wavelengths (DAPI channel) and lowest in the far-red (Cy®5 channel). Our CF®633 bungarotoxin (catalog no. 00009) is a far-red conjugate for the Cy®5 channel with a low negative charge that should have low background from either the dye or autofluorescence.
We test fluorescent bungarotoxin on rat skeletal muscle sections. While the tissue shows autofluorescence, the bungarotoxin staining of motor endplates is usually much brighter than the background for all of the dye colors we’ve tested. However, if you are staining human tissue (especially brain), lipofuscin autofluorescence may be bright in all channels. This usually shows up as bright, punctate dots around cell nuclei. While we would usually recommend our TrueBlack® lipofuscin quenchers for human brain tissue, they are not compatible with bungarotoxin staining. We have, however, found that EverBrite TrueBlack® Mounting Medium (cat. no. 23017) can be used to mount skeletal muscle sections stained with bungarotoxin.
Cy Dye is a registered trademark of Cytiva.
TrueBlack® Lipofuscin quencher can be used with in situ hybridization. There are several references in literature describing this (a few are listed below).
https://doi.org/10.1101/2020.02.03.931618.
https://doi.org/10.1186/s13195-019-0469-0.
If solvents or buffers containing detergents are used, TrueBlack treatment should be performed after these steps, preferably after rinsing the section with PBS. TrueBlack® may also precipitate out due to the high salt SSC wash buffers used. Rinsing the sections in PBS prior to TrueBlack treatment may help minimize this.
TrueBlack® lipofuscin quencher should be mounted using an aqueous-based mounting medium. It cannot be used with organic solvent-based mounting media like Permount™ or DPX.
The TrueBlack® lipofuscin quencher is compatible with the following mounting media (with or without DAPI):
EverBrite™ Mounting Medium
EverBrite™ Hardset Mounting Medium
Slowfade® Gold (Thermo Fisher Scientific)
Prolong® Gold (Thermo Fisher Scientific)
VECTASHIELD® (Vector Laboratories)
VECTASHIELD® Hardset (Vector Laboratories)
Fluoromount-G® (Southern Biotech)
Fluoromount™ (Sigma)
For best results, we recommend mounting samples with a glycerol-based wet-set mounting medium like EveBrite™ Mounting Medium. The quencher is also compatible with EverBrite™ Hardset Mounting Medium.
We’ve also found these mounting media to be compatible for mounting TrueBlack® Plus treated samples.
SlowFade® Gold
SlowFade® Diamond
Prolong® Diamond
Prolong® Glass
Vectashield® Hardset
Vectashield® Vybrance™
Fluoromount-G®
Mowiol®-based mounting medium
We do not recommend using the following mounting media:
Vectashield® wet-set medium
Fluoroshield™
The TrueBlack® lipofuscin quencher is hydrophobic in nature. Certain experimental conditions can cause the quencher to leave precipitates or clumps on the treated sample which can interfere with imaging. We recommend heating the vial of the stock solution of TrueBlack®, 20X in DMF to 70ºC for 5 min. to avoid this. Diluting and storing TrueBlack as a bulk 1X solution in 70% ethanol can also help.
TrueBlack® and TrueBlack® Plus are hydrophobic in nature and quench lipofuscin autofluorescence mainly through hydrophobic interactions. They could therefore stain/bind to lipid structures and quench the fluorescence signal of BODIPY and other lipid droplet stains. Also, original TrueBlack® #23007 is used in 70% EtOH, which could interfere with lipid droplet morphology and affect staining. TrueBlack® Plus #23014, on the other hand, can be used in buffer instead of 70% ethanol, and may be more suitable for combining with lipid droplet staining. However, since the binding of TrueBlack® Plus is also dependent on hydrophobic interactions, we recommend testing TrueBlack® pre-treatment and post-treatment for compatibility with lipid droplet staining in your sample type.
TrueBlack® Plus Lipofuscin Autofluorescence Quencher is cell membrane permeant, and at low concentrations it is not toxic to live cells. In live cells, the dye accumulates in lysosomes and other intracellular vesicles. Therefore, it may or may not be useful for quenching live cell autofluorescence for flow cytometry or microscopy, depending on the source and localization of the autofluorescence and target probes in the specific cell type.
The concentrations of TrueBlack® Plus that we’ve tested in live cells are much lower than those used for quenching in tissue sections, 0.001X to 0.005X (corresponding to 1:40,000 to 1:8,000 dilution of the 40X stock). Incubation can be done in cell culture medium for 30 minutes to several days, though toxicity may vary between cell types.
Our original TrueBlack® Lipofuscin Autofluorescence Quencher (catalog no. 23007) is not soluble in aqueous buffers or media, and therefore is not compatible with live cell staining.