Primary Antibody Conjugates

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.  

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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.

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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.

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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.  

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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

Akoya Tyramide	Ex/Em (nm)	Biotium Tyramide	Ex/Em (nm)	Biotium Cat. No.	Notes	CF® Dye Features
TSA Coumarin	402/443	CF®405S	404/431	92197	Much brighter & more photostable alternative	CF®405S Features
Opal™ Polaris 480	450/500	CF®430	426/498	96053	Recommended for abundant targets	CF®430 Features
Opal™520	494/525	CF®488A	490/515	92171		CF®488A Features
TSA Fluorescein	494/517	Fluorescein	492/514	96018	Direct replacement	See CF®488A for a brighter & much more photostable alternative
TSA TMR TSA Cyanine 3	550/570	Cyanine 555	555/565	96020	Direct replacement for Cyanine 3	See CF®555 & CF®568 for brighter & more photostable alternatives.
		CF®555	555/565	96021	Brighter & more photostable alternatives	CF®555 Features
		CF®568	562/583	92173		CF®568 Features
Opal™570	550/570	CF®550R	551/577	96077		CF®550R Features
		CF®555	555/565	96021		CF®555 Features
TSA Plus Cyanine 3.5	581/596	CF®583R	586/609	96085	Brighter & more photostable alternatives	CF®583R Features
		CF®594	593/614	92174		CF®594 Features
Opal™620	588/616	CF®583R	586/609	96085		CF®583R Features
TSA Cyanine 5	648/667	CF®640R	642/662	92175	Brighter & more photostable alternative	CF®640R Features
		CF®647	650/665	96022	Brighter alternative	CF®647 Features
		CF®660R	663/682	92195	Less cross-talk with visible red dyes; bright & extremely photostable	CF®660R Features
TSA Plus Cyanine 5.5 Opal™690	673/692 676/694	CF®680R	680/701	92196	Bright & extremely photostable	CF®680R Features
Opal™ Polaris 780	750/770	CF®754	745/786	96090	Recommended for abundant targets	Unique NIR dye for TSA
TSA Biotin	N/A	Biotin-XX	N/A	92176	Direct replacement
TSA Plus DNP	N/A	DNP	N/A	96019	Direct replacement
TSA Plus DIG	N/A	N/A	N/A	N/A	N/A

Note: The CF® Dyes listed here are spectrally similar alternatives to Opal™ dyes, they have not been validated as direct replacements for use with Akoya's kits or imaging system. Opal is a trademark of Akoya Biosciences.  

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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.

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