Select Format Without Nuclear Stain With DAPI With NucSpot® 640
Select Size 2 mL 10 mL Request Quote or Bulk Order
🔍
Methanol-fixed human cerebral cortex cryosections were stained with CF® 488A Anti-GFAP, clone GA5+ASTRO/789 (glial cells, green), then mounted with EverBrite™ Hardset or EverBrite TrueBlack® Hardset mounting medium with NucSpot® 640 (nuclei, magenta). Lipofuscin autofluorescence was imaged in the Cy® 3 channel (orange). Sections were imaged after curing overnight at room temperature. EverBriteTrueBlack® Hardset effectively quenched lipofuscin autofluorescence. Methanol-fixed human cerebral corex cryosections mounted in EverBrite™ Hardset with DAPI (top), or EverBrite TrueBlack® Hardset with DAPI (bottom). EverBrite TrueBlack® mounting medium effectively quenches lipofuscin autofluorescence, which appears pink/white in the merge of the green, red, and far-red channels (right). Nuclei are stained blue with DAPI in the merge of all channels (left). Human cerebral cortex cryosections were stained with CF® 405S GFAP Antibody (ASTRO/1974R) (glial cells, cyan), then mounted with EverBrite™ Hardset with NucSpot® 640, or EverBrite TrueBlack® Hardset with NucSpot® 640. Lipofuscin autofluorescence appears orange in the merged image and is effectively quenched after mounting in EverBrite TrueBlack® . Nuclei are stained with NucSpot® 640 (magenta). Methanol-fixed human cerebral cortex cryosections were stained with CF® 488A Anti-GFAP GA5 + ASTRO/789, then mounted with EverBrite™ Hardset Mounting Medium with DAPI or EverBrite TrueBlack® Hardset Mounting Medium with DAPI. Coverslips were allowed to cure overnight at room temperature. Merged images of blue, green, red, and far-red channels are shown. Lipofuscin autofluorescence appears pink/red in the sample mounted with EverBrite™ Hardset (left). EverBrite TrueBlack® Hardset (right) quenched lipofuscin fluorescence while preserving signal from GFAP (green) and DAPI (blue). PFA-fixed rat testis cryosection stained with CF® 488A phalloidin (green) and mounted in EverBrite TrueBlack® Hardset Mounting Medium with NucSpot® 640 (magenta). Photostability of FITC staining in HeLa cells with various mounting media. EverBrite™ Hardset Mounting Medium prolongs FITC fluorescence significantly when compared with ProLong® Gold Antifade Mountant (Thermo Fisher Scientific). Photostability of Cy® 5 staining in HeLa cells with various mounting media. A major advantage of EverBrite™ Hardset Mounting Medium is that, unlike Vectashield® , it does not decrease the fluorescence of cyanine-based fluorophores. Fluorescence values for Cy® 5 in Vectashield® medium are normalized to PBS at time t0 to illustrate the drop in fluorescence of cyanine dyes caused by Vectashield® .
Product Description
This unique hardset antifade mounting medium quenches lipofuscin autofluorescence, for one-step mounting, quenching, and optional nuclear counterstaining of fluorescently-stained tissue sections.
Features
The only mounting medium with autofluorescence quenching
Quenches as it hardens, with low background fluorescence
Optimally formulated for protecting CF® Dyes and other dyes from photobleaching
Refractive index well-matched to coverglass (1.46 after curing)
Choice of DAPI, far-red NucSpot® 640, or without nuclear counterstain
Spectral Properties of Nuclear Stains
DAPI: 358/461 (with DNA)
NucSpot® 640: 649/668 nm (with DNA) (for Cy® 5 channel)
TrueBlack® Lipofuscin Quenchers are designed to quench lipofuscin autofluorescence, a common source of background in human and aged animal tissues such as brain and retina. They also may reduce background from other sources like red blood cells or extracellular matrix. EverBrite TrueBlack® Hardset Mounting Medium performs lipofuscin quenching and mounting in a single step, for greater convenience. The mounting medium substantially reduces lipofuscin fluorescence after mounting, and quenching continues to improve as the medium cures and hardens overnight. Unlike VECTASHIELD® Mounting Medium, EverBrite TrueBlack® is compatible with cyanine-based fluorophores and is optimally formulated for use with Biotium’s CF® Dyes.
Note: EverBrite TrueBlack® Hardset may reduce specific fluorescence; antibody or probe concentration may require optimization for use with the mounting medium.
EverBrite TrueBlack® is available with or without DAPI, or with Biotium's novel NucSpot® nuclear-specific counterstains. NucSpot® 640 is a far-red dye for the Cy® 5 channel. Using a far-red nuclear stain can avoid problems of fluorescence cross-talk and UV photoconversion with traditional dyes like DAPI. See our other NucSpot® Nuclear Stains for bright and specific nuclear counterstaining, available from green to near-IR.
Note: NucSpot® dyes also show dim blue fluorescence with the DAPI filter set, and should be tested for suitability before using with blue probes, especially for epifluorescence microscopy.
Choose the right quencher for your application
Product Catalog no. Pros Cons
TrueBlack® Lipofuscin Autofluorescence Quencher 23007 • Complete quenching of lipofuscin autofluorescence • Introduces some red/far-red background
TrueBlack® Plus Lipofuscin Autofluorescence Quencher 23014 • Quenching performed in PBS or other buffer® • Titration recommended for optimal quenching® for high-lipofuscin samples
EverBrite TrueBlack® Hardset Mounting Medium 23017 • The only mounting medium for one-step mounting and quenching® • May not be as effective as the original TrueBlack® for high-lipofuscin samples® dyes should be tested for suitability with blue dyes
EverBrite TrueBlack® Hardset Mounting Medium with DAPI 23018
EverBrite TrueBlack® Hardset Mounting Medium with NucSpot® 640 23019
See our full selection of antifade mounting media:
Product Nuclear Counterstain Cat. No. Features
EverBrite™ Mounting Medium None 23001 • Wet-set mounting medium
EverBrite™ with DAPI DAPI 23002
Drop-n-Stain EverBrite™ Mounting Medium None 23008 • Wet-set mounting medium
Drop-n-Stain EverBrite™ with DAPI DAPI 23009
EverBrite™ Hardset Mounting Medium None 23003 • Hard-set mounting medium
EverBrite™ Hardset with DAPI DAPI 23004
EverBrite™ Hardset with NucSpot® 640 NucSpot® 640 23016
EverBrite TrueBlack® Hardset Mounting Medium None 23017 • Unique antifade with lipofuscin quenching
EverBrite TrueBlack® Hardset with DAPI DAPI 23018
EverBrite TrueBlack® Hardset with NucSpot® 640 NucSpot® 640 23019
CoverGrip™ Coverslip Sealant N/A 23005 • For sealing edges of wet-set coverslips
* Note: NucSpot® 640 also have dim fluorescence with the DAPI filter set, and should be tested for suitability with blue probes, especially for epifluorescence imaging.
Cy Dye is a registered trademark of Cytiva; VECTASHIELD is a registered trademark of Vector Laboratories.
Technology
Considerations for Immunofluorescence Staining
TrueBlack® Lipofuscin Autofluorescence Quencher
Tech Tips
Immunofluorescence Staining Tips
Tech Tip: Avoiding Artifacts from UV Photoconversion of DAPI and Hoechst
Tech Tip: Battling Tissue Autofluorescence
Troubleshooting Tips for Fluorescence Staining
Protocol: Immunofluorescence Staining of Cells for Microscopy
Tech Tip: Fluorescence Bleed-Through and Cross-Talk
Product Attributes
For live or fixed cells
For fixed cells/tissue sections
Probe cellular localization
Nucleus
Excitation/Emission
DAPI: 358/461 nm (with DNA), NucSpot® 640: 649/668 nm (with DNA)
Storage Conditions
Store at 2 to 8 °C, Protect from light
Documents, Protocols, SDS and COA
Datasheet
Datasheet Download
COA Request the Certificate of Analysis
You might also be interested by the following products:
