FAQs

See below for our recommended alternatives to Akoya tyramides. Unless noted as a direct replacement, these tyramide dyes are spectrally similar alternatives. They have not been validated as direct replacements for use with Akoya kits or imaging systems.

CF® Dye Alternatives for Akoya Tyramides

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF® Dye succinimidyl ester stability

Succinimidyl esters (SE) are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Many commercially available fluorescent dyes used for life science research are heavily sulfonated dyes which makes them particularly hygroscopic, worsening the hydrolysis problem. In addition, for several commercially available SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while the SE group in all of Biotium’s CF® Dyes is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF® Dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

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.

Cy Dye is a registered trademark of Cytiva.

See our CF® Dye Quick Reference Table for a list of dyes and summary of their features. Our CF® Dye Selection Guide has more detailed information on each CF® dye, and ordering information for our various CF® dye product lines. You can download CF® dye normalized absorption and emission data in Excel format.

The exact chemical structures of CF® dyes are currently confidential but will be fully disclosed at a later stage when pending patents become granted. In general terms, the structure of a CF® dye may be divided into two parts: a) dye core structure (i.e. the aromatic ring skeleton that defines the dye’s color or absorption/emission wavelengths), and b) core structure-modifying elements. At present, CF® dyes bear the core structures of coumarin, pyrene, rhodamine or cyanine dyes. Blue fluorescent CF® dyes are based on coumarin or pyrene dye core structure, while green to near-IR CF® dyes are based on either cyanine or rhodamine dye core structures. Core structure-modifying elements refer to various chemical attachments to the core structure and are a key aspect of the CF® dye invention that makes CF® dyes superior to other commercial dyes.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature or with an ice pack. These products may thaw without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Some products are shipped with blue ice packs as an extra precaution against high temperatures. The blue ice packs may be thawed upon arrival without affecting product performance.

Products with recommended storage at ultra low temperature (-70°C) that also ship on dry ice should arrive frozen. If a product you received was shipped on dry ice and thawed during transit, please contact customer service at order@biotium.com.

You can also download our Product Storage Statement here.

Most CF® dyes carry 1-2 negative charges while a few cyanine-based near-IR CF® dyes carry 3-4 negative charges. However, the more negatively charged CF® dyes comprise unique structural features that shield the negative charges such that the biomolecules (such as antibodies) the dyes label do not lose specificity due to the excessive negative charges.

CF® initially was an abbreviation for Cyanine-based Fluorescent dyes. These were the first patented CF® Dyes based on cyanine dye structures. Since then, our CF® Dye patent portfolio has expanded to include four different fluorescent dye core structures that cover the fluorescence spectrum from UV to NIR. Today, we believe “CF” more aptly stands for Clear Fluor: dyes that produce superior signal-to-noise.

CF® dyes are highly water soluble (>100 mg/mL). They are also very soluble in other polar solvents, such as DMSO, DMF, methanol and ethanol. However, CF® dyes are poorly soluble or insoluble in non-polar solvents.

Rhodamine-based CF® dyes (designated R) generally have better photostability but weaker fluorescence than their cyanine-based equivalents (designated C). Therefore, rhodamine-based near-IR CF® dyes are a better choice for microscopy, while cyanine-based CF® dyes are more ideal for flow cytometry, Western blotting, and other applications where photobleaching is less of a concern. Another factor to consider is the size of the dyes. Some of the cyanine-based near-IR CF® dyes are much larger than the rhodamine-based equivalents. For antibody labeling, either version of the CF® dyes is suitable. However, for applications where the dye size may cause a steric problem, the smaller dye may be a better choice.

We don’t have direct experience or customer feedback on CF dyes for 6-color imaging on LSM 800, but because this instrument has tunable excitation and linear unmixing, you should have a lot of flexibility in dye choice. We would also recommend contacting Zeiss support to see if they have advice for selecting dyes for multi-color experiments for the instrument.

The violet excited 405/430 dyes are generally less bright and should be used for more abundant targets. The 754 dye would also be recommended for more abundant targets. Even with optimal excitation, the GaAsp detectors on the Zeiss LSM 880 are expected to be less responsive to NIR wavelengths. The other dyes are very bright and would be better for less abundant targets to minimize cross-talk.

Below we’ve listed the dyes we’d usually recommend for the standard laser lines on the LSM 880.

405 nm laser line

CF®405S, Ex/Em 404/431 nm; recommended for abundant targets

458 nm laser line

CF®430, Ex/Em 426/498 nm; recommended for abundant targets

488 nm laser line

CF®488A, Ex/Em 490/515 nm; bright and photostable

Note: CF®488A and CF®514 must be separated by spectral imaging if used together

514 nm laser line       

CF®514, Ex/Em 516/548 nm; dye for spectral imaging

Note: CF®488A and CF®514 must be separated by spectral imaging if used together

561 nm laser line

CF®568 Ex/Em 562/583 nm; very bright and photostable

Note: CF®568 and CF®594 must be separated by spectral imaging if used together

594 nm laser line

CF®594, Ex/Em: 593/614 nm; very bright and photostable

Note: CF®568 and CF®594 must be separated by spectral imaging if used together

633 nm laser line

CF®640R, Ex/Em 642/662 nm; CF®640 will be brighter with on-center excitation

CF®660R, Ex/Em 663/682 nm; CF®660R is off-center for this laser, but very bright and extremely photostable; less likely to have cross-talk with CF®594

Tunable 2P Laser 720-1020

CF®754, Ex/Em 745/786; recommended for abundant targets (LSM800 detectors are expected to be less sensitive for NIR dyes)

All three of these dyes can be excited by the 405 nm laser (or UV mercury lamp). They differ in their emission wavelengths. CF®405S has blue fluorescence emission at 431 nm, similar to AlexaFluor® 405, Cascade Blue®, and DyLight™ 405 (Thermo Scientific). CF®405M has longer wavelength blue fluorescence emission at 452 nm, similar to Horizon™ V450 (BD Biosciences) and Pacific Blue® (Thermo Scientific). CF®405L has orange fluorescence emission at 545 nm, similar to Pacific Orange® (Thermo Scientific). We recommend choosing the dye that best fits your instrument’s detection settings. See our CF® Dye Quick Reference Table and CF® Dye Selection Guide for more information on CF® dyes.

There are usually three aspects to dye stability: 1) chemical stability of the dye core structure; 2) stability of the reactive group; and 3) photostability of the dye.

Chemical stability of the dye core structure:

This refers to resistance of the dye core structure to decomposition caused by factors other than photo-bleaching. These factors may include temperature, pH and incompatibility with other chemicals in the medium. This type of stability information is most useful for estimating the shelf-life of the dye that is already covalently attached to another molecule (e.g., an antibody), or for assessing the chemical compatibility of the dye in certain applications. CF® Dyes bear the core structures of coumarin, pyrene, rhodamine or cyanine dyes, all of which are known to have excellent chemical stability. In general, CF® Dyes are far more stable than the antibodies they label. Thus, if a CF®-labeled antibody loses activity during storage, the problem is not likely to be caused by the dye. CF® Dyes are also stable enough for labeled nucleic acids to be used in PCR or nucleic acid hybridization, where high temperature is involved.

Stability of the reactive group:

Reactive CF® Dyes comprise a reactive group used in bioconjugation. Among the various reactive groups, only amine-reactive succinimidyl ester (SE) and thiol-reactive maleimide groups are unstable because the small amount of moisture trapped in or leaked into the packaging vials can cause hydrolysis of the reactive groups over time. The SE group, in particular, is susceptible to degradation. Thus, in order to slow degradation, CF® Dyes comprising these reactive groups must be stored at -20°C under anhydrous conditions. Furthermore, stock solutions of the dyes must be made using dry solvents, such as anhydrous DMSO. One advantage of CF® Dye SE products over other commercial dyes is their relatively high stability. Normally, an SE group can be derived from either an aliphatic or an aromatic carboxylic acid group, but an aliphatic carboxylic group tends to result in a more stable SE, offering higher resistance to hydrolysis and thus better labeling efficiency. All of the CF® SE Dyes have their SE groups derived from aliphatic carboxylic acid groups, unlike many commercially available SE dyes that are often prepared from aromatic carboxylic acid groups.

Photostability:

This refers to the dye’s ability to withstand photobleaching. For most dyes, photostability is not a major problem for routine handling under ambient light or for applications, such as flow cytometry and Western blotting, where the dyes are only briefly exposed to light. However, for microscopy, especially for confocal microscopy, where the dyes may be subject to intense illumination for an extended period of time, photobleaching can be a major concern. Similar to the photostability of other fluorescent dyes, both the dye core structure and the structure-modifying groups attached to it play a role in the photostability of CF® Dyes. CF® Dyes bear the core structure of rhodamine, cyanine, pyrene or coumarin dyes; among the four types of core structures, rhodamine core is the most photostable, followed by cyanine and then by pyrene and coumarin cores. The structure-modifying groups and the way they are attached to the dye cores are a key innovative aspect of CF® Dye technologies that contributes to the superior photostability of CF® Dyes over that of other commercial dyes. In general, rhodamine-based CF® Dyes, whose wavelengths range from visible to the near-IR region, offer the best photostability, making the dye ideal for microscopy applications.

CF® dyes are chemically stable within the pH range of at least 2 –11. The fluorescence of most CF® dyes is relatively insensitive to pH, except for that of CF®405M, CF®568, CF®620R, and CF®633. The fluorescence of these four CF® dyes becomes weaker when pH drops below 4.5.

CF® dyes can tolerate formaldehyde fixation. However, whether a CF® dye-labeled probe is fixable will depend on the fixability of the probe itself. Proteins with free amine groups that bind other proteins generally are formaldehyde-fixable.

There is no simple answer to this question as the quantum yield of a fluorescent dye can vary widely, depending on the dye’s micro-environment. For example, the quantum yield of a dye attached to a protein may be very different from the quantum yield of the free dye. For dyes attached to a protein, the quantum yield is highly dependent on how many molecules of the dye are attached to the protein (i.e. degree of protein labeling). In general, a higher degree of protein labeling leads to a lower dye quantum yield due to fluorescence quenching via dye-to-dye interaction. For this reason, as the degree of labeling increases, fluorescence intensity of the labeled protein will eventually reach a maximum and start to decline thereafter. In fact, one of the best ways to compare the relative quantum yields of different dyes is to plot the total fluorescence of the labeled proteins as a function of degree of labeling by the dyes as we have done with CF® dyes and other commercial dyes. CF® dyes generally give higher slopes than other commercial dyes in the plots, suggesting less quantum yield decline with increasing degree of protein labeling.

Lifetime data for several CF® dyes are available, both for the free dye in buffer as well as for the corresponding CF® dye conjugated secondary antibody. These are listed below.

Measurements were made on a Stellaris 8 STED FALCON microscope courtesy Leica Microsystems, Germany. § Fluorescence lifetime measurements of CF® dye labeled anti-mouse secondary antibodies used for immunostaining microtubules in U2OS using mouse anti-tubulin (DM1a) and mounted in ProLong™ Diamond. *Lifetime data obtained via customer communication under different experimental conditions and imaging setup.

CF® dyes are ideal for protein labeling because of their high water solubility, which reduces fluorescence quenching. They are also useful for labeling oligonucleotides that require multiple copies of a dye for maximal fluorescence, such as the preparation of FISH probes, where water soluble dyes can minimize fluorescence quenching. Finally, CF® dyes make excellent polar tracers that can be used for visualizing the morphology or long-term tracing of neurons after microinjection. CF® dyes and their conjugates are ideal for fluorescence microscopy applications, flow cytometry, Western blotting, and in vivo imaging (near-IR CF® dyes).

Reactive dyes for bioorthoganol conjugation (click chemistry) including alkyne, azide, BCN, TCO, and methyltetrazine can be used to prepare polymers. We do not offer CF dye methacrylate at this time, please contact techsupport@biotium.com to inquire about custom synthesis.

Yes, CF® dye tyramides can be used for in situ hybridization using Tyramide Signal Amplification¹ (TSA). They are also compatible with the RNAscope assays  for RISH².

¹ doi: https://doi.org/10.1038/s41598-018-38171-5.

² doi: http://dx.doi.org/10.1101/651083.

If you order an oligo to be synthesized with an amino group on one end, then our CF® Dye succinimidyl ester (SE) dyes could be used to label it using a standard amine labeling protocol for DNA.

We recommend using an HPLC purified oligo, with an amino group with a C6 linker at the 5′ or 3′ end. To remove free dye after labeling, you could use G25 Sephadex, gel purification, ethanol precipitation, or HPLC, depending on the degree of purity needed.

Alternatively, this may be something that an oligo synthesis company may be able to perform for you as a custom labeling.

We do not have firsthand experience with LPS labeling, but according to the literature,  LPS has been labeled using amine-reactive dyes, like FITC. Our amine-reactive CF® Dye Succinimidyl Esters should also work for this. There is a publication for enzymatic labeling of LPS using dye hydrazides. Our CF® Dye hydrazides could be used in this method. The paper also describes the traditional amine labeling method and purification of the conjugate.

For dyes or reagents that are supplied lyophilized (as solids), it is hard to compare quantities based on appearance of the dye in the tube, because during the lyophilization process the dye can dry down in different ways, either spread out all over the tube, clumped together, or coating the sides or bottom of the tube. Centrifugation of the tube may not help in collecting the dye solid to the bottom of the tube as this generally works for solutions. However, lyophilized solids are packaged based on highly accurate absorbance measurement of the reagent solution prior to drying, so the vial will contain the correct amount of dye.