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 |
|Opal™ Polaris 480||450/500||CF®430||426/498||96053||Recommended |
for abundant targets
|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 |
|TSA Plus Cyanine 3.5||581/596||CF®583R||586/609||96085||Brighter & more |
|TSA Cyanine 5||648/667||CF®640R||642/662||92175||Brighter & more |
|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
|TSA Plus Cyanine 5.5|
|CF®680R||680/701||92196||Bright & extremely|
|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|
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: https://doi.org/10.1038/s41598-018-38171-5.
2 doi: http://dx.doi.org/10.1101/651083.
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.