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.
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.
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).