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Tyramide Signal Amplification (CF® Dye & Other Tyramides)

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 TyramideEx/Em (nm)Biotium TyramideEx/Em (nm)Biotium Cat. No.NotesCF® Dye Features
TSA Coumarin402/443CF®405S404/43192197Much brighter & more
photostable alternative
CF®405S Features
Opal™ Polaris 480450/500CF®430426/49896053Recommended
for abundant targets
CF®430 Features
Opal™520494/525CF®488A490/51592171CF®488A Features
TSA Fluorescein494/517Fluorescein492/51496018Direct replacementSee CF®488A for a brighter &
much more photostable alternative
TSA Cyanine 3
550/570Cyanine 555555/56596020Direct replacement
for Cyanine 3
See CF®555 & CF®568 for brighter
& more photostable alternatives.
CF®555555/56596021Brighter & more
photostable alternatives
CF®555 Features
CF®568562/58392173CF®568 Features
Opal™570550/570CF®550R551/57796077CF®550R Features
96021CF®555 Features
TSA Plus Cyanine 3.5581/596CF®583R586/60996085Brighter & more
photostable alternatives
CF®583R Features
CF®594593/61492174CF®594 Features
Opal™620588/616CF®583R586/60996085CF®583R Features
TSA Cyanine 5648/667CF®640R642/66292175Brighter & more
photostable alternative
CF®640R Features
CF®647650/665 96022Brighter alternativeCF®647 Features
CF®660R663/68292195Less cross-talk with visible red dyes;
bright & extremely photostable
CF®660R Features
TSA Plus Cyanine 5.5
CF®680R680/70192196Bright & extremely
CF®680R Features
Opal™ Polaris 780750/770CF®754745/78696090Recommended
for abundant targets
Unique NIR dye for TSA
TSA BiotinN/ABiotin-XXN/A92176Direct replacement
TSA Plus DNPN/ADNPN/A96019Direct replacement
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.


Our Tyramide Amplification Kits have been demonstrated to be robust and versatile for multi-color fluorescence imaging, compatible with dye-labeled antibodies and various cell staining methods (see Figure 1).

To use a Tyramide Amplification Kit in addition to one or more dye-labeled antibodies, follow the kit protocol to fix and block samples; label with primary antibodies; then detect primary antibodies using secondary antibodies. Dye labeled secondary antibodies can be co-incubated with the HRP-conjugated secondary or HRP-streptavidin from the tyramide kit. After washing, perform the CF® Dye tyramide reaction according to the kit protocol. The tyramide reaction does not interfere with the binding of dye-labeled antibodies or other fluorescent staining reagents.

Performing multi-color detection with more than one dye tyramide on the same sample requires sequential tyramide staining reactions, followed by HRP inactivation or antibody stripping between each step. See our tech tip:

Multi-Color Fluorescence Imaging Using Biotium’s Tyramide Amplification Kits

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:

2 doi:

We do not have any firsthand information about whether increasing tyramide incubation time will improve staining for thicker sections. We have tested tyramide on slide-mounted cryosections that are 10 um thick. Published protocols for 40 um floating sections recommend performing the tyramide development step for 15 minutes at room temperature.

If you are using free-floating sections, generally all incubation steps (permeabilization, antibody incubation, and washing) are longer than for sections on slides. The tyramides are small molecules and should penetrate tissue more rapidly than antibody conjugates, but you may wish to test a longer incubation time to allow the buffer to penetrate the tissue.

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