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.
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.
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® Plus is a next-generation lipofuscin quencher developed by Biotium chemists. TrueBlack® Plus is water-soluble, so quenching can be performed in PBS instead of ethanol. It greatly reduces lipofuscin autofluorescence with minimal far-red background.
Quenches lipofuscin with lower far-red background than our original TrueBlack® (23007)
The only lipofuscin quencher that can be used in aqueous buffer instead of 70% EtOH
Quenching in buffer allows longer incubation times for thick samples without shrinkage, is compatible with hydrophobic stains.
No hazardous solvents
Reduces autofluorescence from other sources like red blood cells and extracellular matrix
Treat tissues before or after immunofluorescence staining
TrueBlack® Plus: A unique lipofuscin quencher
Autofluorescence is a major source of non-specific background fluorescence in tissue sections and some primary cell types. Sources of autofluorescence include aldehyde fixatives, tissue components with endogenous fluorescence (including extracellular matrix proteins, red blood cells, and macrophages), and lipofuscin, which consists of highly autofluorescent granules of oxidized proteins and lipids that build up in the lysosomes of cells with age. While usually brightest in the blue and green wavelengths, autofluorescence has broad spectrum fluorescence that can make detection of specific fluorescence signal in tissues virtually impossible unless it is quenched or masked.
Many treatments have been reported to reduce autofluorescence, including quenching of aldehydes with ammonium sulfate and Tris, bleaching with sodium borohydride, and quenching of autofluorescence with blue or black dyes. The lipophilic dye Sudan Black B is highly effective at masking autofluorescence from lipofuscin, but has the drawback of introducing red fluorescent background. Our original TrueBlack® Lipofuscin Autofluorescence Quencher (cat. no. 23007) was developed as an alternative to Sudan Black B. It effectively quenches lipofuscin autofluorescence with much lower background than Sudan Black B, but still introduces a low level of far-red background. Also, like Sudan Black B, treatment with original TrueBlack® quencher must be performed in 70% ethanol, which is incompatible with some staining protocols and specimen types.
TrueBlack® Plus was developed to allow lipofuscin quenching in aqueous buffer with even lower background than original TrueBlack®. Quenching in PBS allows longer incubation times for thick samples without shrinkage, and is compatible with hydrophobic stains.
Fluorescent CF® Dye tyramides are used for tyramide signal amplification (TSA) for increasing immunofluorescence sensitivity in multicolor immunocytochemistry (ICC), immunohistochemistry (IHC), or in situ hybridization (ISH).