iFluor® 555 Tyramide

For many immunohistochemical (IHC) applications, traditional enzymatic amplification procedures are sufficient for achieving adequate antigen detection. However, several factors limit their sensitivity and utility. Tyramide signal amplification (TSA) has proven to be a particularly versatile and powerful enzyme amplification technique with improved assay sensitivity. TSA is based on the ability of HRP, in the presence of low concentrations of hydrogen peroxide, to convert labeled tyramine-containing substrate into an oxidized, highly reactive free radical that can covalently bind to tyrosine residues at or near the HRP. To achieve maximal IHC detection, tyramine is prelabeled with a fluorophore. The signal amplification conferred by the turnover of multiple tyramide substrates per peroxidase label results in the ability to detect low-abundance targets with ultrasensitive precision and reduces the amount of antibodies and hybridization probes needed. In IHC applications, this method can also enhance sensitivity in cases where the primary antibody dilution needs to be increased to reduce nonspecific background signals or overcome weak immunolabeling due to suboptimal fixation procedures or low levels of target expression. The iFluor® 555 tyramide contains the bright iFluor® 555 that can be readily detected with the standard Cy3 filter set. It is an excellent replacement for Alexa Fluor® 555 tyramide (Alexa Fluor® is the trademark of ThermoFisher), Cy3 tyramide, or other comparable fluorescent tyramide conjugates.

Formalin-fixed, paraffin-embedded (FFPE) human lung tissue was labeled with anti-EpCAM mouse mAb followed by HRP-labeled goat anti-mouse IgG (Cat No. 16728). The fluorescence signal was developed using iFluor® 555 tyramide (Cat No. 45105) and detected with a TRITC/Cy3 filter set. Nuclei (blue) were counterstained with DAPI (Cat No. 17507).

Protocol

SDS

COA

Catalog	Size	Price	Quantity
45105	200 Slides	Price

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® 555 Tyramide to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	95.565 µL	477.824 µL	955.648 µL	4.778 mL	9.556 mL
5 mM	19.113 µL	95.565 µL	191.13 µL	955.648 µL	1.911 mL
10 mM	9.556 µL	47.782 µL	95.565 µL	477.824 µL	955.648 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
mg	÷	1046.41 g/mol	=	mL	x	mM	=	mmol

Physical properties

Molecular weight	1046.41
Solvent	DMSO

Spectral properties

Correction factor (260 nm)	0.23
Correction factor (280 nm)	0.14
Extinction coefficient (cm ^-1 M ^-1)	100000 ¹
Excitation (nm)	557
Emission (nm)	570
Quantum yield	0.64 ¹

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Instrument settings

Fluorescence microscope
Excitation	Cy3/TRITC filter set
Emission	Cy3/TRITC filter set
Recommended plate	Black wall/clear bottom

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Page updated on October 9, 2025