TrueBlack® Lipofuscin Autofluorescence Quencher, 20X in DMF, is a new reagent for quenching lipofuscin autofluorescence in tissue sections for immunofluorescence staining. It can also help reduce autofluorescence from other sources like extracellular matrix.
The storage conditions that a Biotium label recommends are deliberately conservative. Long term storage at low temperatures usually will increase the time that the product will remain within specification, unless specific instructions otherwise are provided (such as “do not refrigerate” or “do not freeze”).
Biotium labels and product information sheets may show a specific temperature for long term storage, or a temperature range that is adequate for long term storage. For all products, the implied range is as follows for the following storage conditions:
For Biotium products where the label indicates room temperature or RT, this implies storage in ambient conditions between 20°C and 30°C.
For Biotium products where the label indicates 4°C, this implies storage in a refrigerator that is between 2°C and 8°C.
For Biotium products where the label indicates -20°C, this implies storage in a freezer that is between -35°C and -5°C.
Ultra Low Temperature Freezer
For Biotium products where the label indicates -70°C, this implies storage in an ultra low-temperature (ULT) freezer that is below -60°C.
Compound solubility is listed on product web pages, product information sheets or product safety and data sheets. Check the product information sheet for specific instructions for preparing stock solutions. Stock solutions are generally prepared at 10X-1000X the final required working concentration, typically in the range of 1-100 mg/mL. For dyes and indicators, we generally prepare a concentrated stock solution at 5-10 mM in the recommended solvent. The dye can then be diluted to the final desired concentration in the buffer or medium used for the application.
> 1000 mg/mL
Very Slightly Soluble
< 0.1 mg/mL
To dissolve lyophilized compounds, simply add the appropriate volume of the recommended solvent to the vial to make the desired concentration stock solution, and swirl or gently vortex to mix. Make sure the solvent comes in contact with the inside walls of the vial to fully recover the product.
Difficult to dissolve compounds can be heated to 50°C or higher (note: we do not recommend heating coelenterazine solutions), and vortexed or sonicated until completely dissolved. Some chemical compounds are just kinetically slow to dissolve. This can be especially true when the compounds are highly pure and in crystalline form. An alternative and gentler way to dissolve these compounds can be to simply rock the compound/solvent mixture in the dark overnight.
When preparing stock solutions in organic solvents such as DMF, DMSO, or alcohol for use in living cells or organisms, make a concentrated stock so that the final concentration of solvent in the working solution will not be toxic. A general guideline for immortalized cell lines is to keep the final solvent concentration below 1%; certain cell lines, primary cells or experimental organisms may be more sensitive to solvent toxicity. Similarly, for enzymatic reactions, the final concentration of solvent should be kept below 1% to avoid inhibiting enzyme activity.
Compounds dissolved in aqueous solution can be sterilized by filtration if necessary. Generally, concentrated stock solutions of compounds in organic solvents do not require sterilization, but culture medium can be sterilized by filtration after addition of the compound.
Some of our large volume light-sensitive products are packaged in amber bottles or amber glass vials, which are easy to handle and protect the products from light. However, amber micro packaging vials (0.5 mL or 2 mL) make it very difficult to see small quantities of dye when preparing solutions or pipetting. Therefore, we only package photoreactive dyes like PMA or PMAxx™ in amber micro packaging vials. Some of our reactive dyes are packaged in transparent vials, then sealed inside a moisture-resistant foil bag, which also will protect them from light.
Other fluorescent dyes in transparent vials should stored in the dark for long term storage. It’s fine to store dye vials uncovered in a windowless refrigerator or freezer with an automatic light shut-off. If dyes will be stored in a glass-front (deli-style) refrigerator, walk-in refrigerator/freezer, or at room temperature with constant light exposure, they should stored in a non-transparent box (like a white cardboard freezer box), in a closed drawer, in a black plastic bag, or covered with aluminum foil.
Fluorescent dyes generally are not sensitive to brief light exposure while they are being handled on the bench during an experiment. To be on the safe side, we usually loosely cover tube racks with a piece of foil if the dye vials are going to be out on the bench for more than 30 minutes or so. But most dyes are stable enough that even accidentally leaving them on the bench for a day will not affect performance. When handling photoreactive dyes like EMA, PMA, or PMAxx, we take the extra precaution of dimming the lights.
Reduces autofluorescence from non-lipofuscin sources
Doesn’t cause high background, unlike Sudan Black B
Can be used before or after immunofluorescence staining
Clears the way for fluorescence imaging of human and aged animal tissues
TrueBlack® Lipofuscin Autofluorescence Quencher, 20X in DMF, is a new reagent for quenching lipofuscin autofluorescence in tissue sections for immunofluorescence staining. Lipofuscin consists of highly autofluorescent granules of oxidized proteins and lipids that build up in the lysosomes of aging cells. Lipofuscin granules fluoresce brightly in all channels used for fluorescence microscopy, and accumulate in a variety of cell and tissue types with age. Consequently, imaging of specific immunofluorescence signal in certain adult human tissues or aged animal tissues can be virtually impossible unless methods are employed to quench or mask lipofuscin fluorescence.
Traditionally, Sudan Black B has been used to quench lipofuscin autofluorescence by incubating tissue sections with the dye after immunofluorescence staining. However, while it masks the autofluorescence from lipofuscin, Sudan Black B also introduces uniform non-specific background fluorescence in the red and far-red channels (Fig. 1), limiting the use of fluorescent dyes in those wavelengths. Now Biotium has developed TrueBlack® as a superior alternative to Sudan Black B. TrueBlack® quenches lipofuscin fluorescence with far less increase in red/far-red background fluorescence (Fig. 1). TrueBlack® treatment can be performed before or after immunostaining. It is rapid, simple, and has minimal effect on signal from fluorescent antibodies or nuclear counterstains, thus preserving the signal-to-noise ratio of the immunostaining (Figs. 2-3).
TrueBlack® effectively eliminates lipofuscin autofluorescence in tissues like human and mouse brain and retina (see Figs. 1-3 and TrueBlack® References). TrueBlack® also can reduce autofluorescence from other sources, such as collagen, elastin, red blood cells, and general background fluorescence. It is not as effective at quenching these sources of autofluorescence as it is for lipofuscin, but it can improve background in human and non-human tissue types (Figs. 4-5). TrueBlack® also has been used to quench background fluorescence on polycarbonate filters used as cell supports for imaging cytometry.
A revolutionary antifade mounting medium optimally formulated for preserving fluorescence of our CF® dyes and other fluorochromes. EverBrite™ Hardset mounting medium hardens at room temperature completely in 24 hours.
CoverGrip™ Coverslip Sealant is the first product specifically designed for sealing the edges of wet-mounted coverslips for fluorescence microscopy. Unlike nail polish, CoverGrip™ contains no ingredients that can leach into aqueous mounting medium and affect specimen fluorescence.
RedDot™2 is a far-red cell membrane-impermeable nuclear dye similar to Draq7™, with excitation and emission at 665/695 nm. The dye is ideal for specifically staining the nuclei of dead cells with minimal cytoplasmic RNA staining.
The SuperHT PAP Pen draws a water repellent circle around slide mounted tissue and prevents the waste of valuable reagents by keeping the liquid pooled in a single droplet. Available in two sizes, regular (4 mm tip) and mini (2.5 mm tip).
Drop-n-Stain™ CF® dye goat anti-mouse IgG (H+L) is provided in a convenient dropper bottle for easy dispensing without pipetting. To minimize cross-reactivity, the antibody has been adsorbed against bovine, horse, human, rabbit and swine serum.