Support & Resources



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 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 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
CF®680R 680/701 92196 Bright & extremely
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
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.

View more FAQs