Fluorescein Tyramide

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

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.

Biotium developed TyraMax™ Amplification Dyes as high-performance TSA dyes, offering brighter, more photostable signals than Aluora® and Opal® reagents. They also remain stable in amplification buffer for up to 24 hours, facilitating automated staining workflows. TyraMax™ Dyes are offered as standalone dye solutions, as 3-color or 5-color dye sets plus DAPI counterstain, and in a sampler for custom panel optimization. See below for alternatives to specific Aluora® and Opal® reagents. Learn more about our reagents for tyramide signal amplification (TSA).

TyraMax™ Amplification Dyes

TyraMax™ 3-Plex Amplification Dye Set with DAPI (Cat. No. 33029)

TyraMax™ 5-Plex Amplification Dye Set with DAPI (Cat. No. 33030)

TyraMax™ Amplification Dye Spectral Sampler (Cat. No. 33031)

Aluora is a registered trademark of Thermo Fisher Scientific; Opal is a registered trademark of Akoya Biosciences.