EverBrite TrueBlack® Hardset Mounting Medium

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).

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.

EverBrite™ Mounting Medium (23001) and EverBrite™ Mounting Medium with DAPI (23002) both have a refractive index of 1.46.  EverBrite™ Hardset Mounting Medium (23003) and EverBrite™ Hardset Mounting Medium with DAPI (23004) both have an initial refractive index of 1.38, which increases to 1.42 after 24 hours of curing, and 1.46 after 4 days, after which it remains constant

Yes, EverBrite™ mounting media are compatible with a wide range of dyes. Unlike VectaShield®, EverBrite™ won’t interfere with cyanine-based fluorophores like Alexa Fluor® 647, Cy®3, or Cy®5.

AlexaFluor is a registered trademark of Thermo Fisher Scientific; Cy Dye is a registered trademark of Cytiva; VectaShield is a registered trademark of Vector Laboratories.

EverBrite™ wet-set mounting medium is an aqueous, glycerol-containing medium. It can be removed from samples by washing several times with PBS or other buffer. However, washing with PBS will not remove DAPI staining.

For coverslips mounted on slides with EverBrite™ Hardset or EverBrite™ wet-set medium, the slides can be soaked in PBS in a slide jar for several hours with gentle agitation until the coverslip slides off (avoid prying or pulling the coverslip, which could damage the sample). Then wash the slide several times to remove any remaining mounting medium. If the coverslips have been sealed with CoverGrip™ Coverslip Sealant, soaking in PBS will remove the sealant as well. Other methods may be required to remove other types of coverslip sealants.