Cy7 tyramide is a red fluorescent dye used as a reporter fluorescent substrate for horseradish peroxidase (HRP)-catalyzed deposition, a signal amplification technique in immunoassays and nucleic acid in situ hybridization.

Physicochemical Properties

Molecular Formula	C49H66N4O8S2
Molecular Weight	903.20
Exact Mass	902.432207
PubChem CID	169494300
Appearance	Dark green to black solid powder
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	17
Heavy Atom Count	63
Complexity	1740
Defined Atom Stereocenter Count	0
SMILES	CCN\1C2=C(C=C(C=C2)S(=O)(=O)[O-])C(/C1=C\C=C\C=C\C=C\C3=[N+](C4=C(C3(C)C)C=C(C=C4)S(=O)(=O)[O-])CCCCCC(=O)NCCC5=CC=C(C=C5)O)(C)C.CC[NH+](CC)CC InChIKey: PKVPZSGTNHZHDH-UHFFFAOYSA-N
InChi Key	PKVPZSGTNHZHDH-UHFFFAOYSA-N
InChi Code	InChI=1S/C43H51N3O8S2.C6H15N/c1-6-45-37-24-22-33(55(49,50)51)29-35(37)42(2,3)39(45)15-11-8-7-9-12-16-40-43(4,5)36-30-34(56(52,53)54)23-25-38(36)46(40)28-14-10-13-17-41(48)44-27-26-31-18-20-32(47)21-19-31;1-4-7(5-2)6-3/h7-9,11-12,15-16,18-25,29-30H,6,10,13-14,17,26-28H2,1-5H3,(H3-,44,47,48,49,50,51,52,53,54);4-6H2,1-3H3
Chemical Name	(2E)-1-ethyl-2-[(2E,4E,6E)-7-[1-[6-[2-(4-hydroxyphenyl)ethylamino]-6-oxohexyl]-3,3-dimethyl-5-sulfonatoindol-1-ium-2-yl]hepta-2,4,6-trienylidene]-3,3-dimethylindole-5-sulfonate;triethylazanium
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Fluorescent Dye
ln Vitro	Experimental Protocol for Cy3 Tyramide Signal Amplification (TSA) Detection Principle: This method is based on the tyramide signal amplification technique, which utilizes horseradish peroxidase to catalyze the tyramide substrate labeled with the Cy7 fluorophore. This process generates a high-intensity, covalently bound fluorescent signal at the target site, thereby enabling highly sensitive detection of the target protein. Experimental Steps: 1. Cell Fixation Fix the cells using a 1x PBS solution containing 3.7% paraformaldehyde at room temperature for 20 minutes. After fixation, rinse the cells twice with 1x PBS solution to thoroughly remove the fixative. 2. Cell Permeabilization Permeabilize the cells using a 0.1% Triton X-100 solution at room temperature for 1 to 5 minutes (the specific duration should be optimized based on the cell type). After permeabilization, rinse the cells twice with 1x PBS solution. 3. Blocking Endogenous Peroxidase Activity Incubate with a 1x PBS solution containing 3% H₂O₂ at room temperature for 10 minutes to quench endogenous peroxidase activity within the cells. After completion, rinse the cells twice with 1x PBS solution. 4. Blocking Non-Specific Sites Use a 1x PBS solution containing 1% bovine serum albumin as a blocking buffer and incubate at 4°C for 30 minutes to block non-specific binding. 5. Primary Antibody Incubation Remove the blocking buffer and directly add an appropriate amount of the primary antibody working solution (diluted using PBS containing 1% BSA). Incubation conditions: can be incubated at room temperature for 1 hour, or at 4°C overnight. After incubation, wash the cells three times with 1x PBS solution, for 5 minutes each time. 6. Secondary Antibody-HRP Incubation Add 100 µL of the horseradish peroxidase-labeled secondary antibody working solution and incubate at room temperature, protected from light, for 1 hour. After incubation, wash the cells three times with 1x PBS solution, for 5 minutes each time. 7. Cy3 Tyramide Signal Amplification and Detection Add 100 µL of the pre-optimized concentration of Cy7 tyramide working solution and incubate at room temperature, protected from light, for 5 to 10 minutes. After incubation, thoroughly rinse the cells three times with 1x PBS solution, for 5 minutes each time, to remove unreacted tyramide substrate. 8. Imaging Use a fluorescence microscope equipped with filter sets suitable for the Cy7 fluorophore (typically excitation/emission wavelengths around ~750/~770 nm) for image acquisition. Notes: Critical Optimization Step: The optimal concentration of the Cy7 tyramide working solution is crucial for the signal-to-noise ratio. It is recommended to precisely optimize it based on the specific experimental system (e.g., cell type, primary antibody) through preliminary experiments (such as concentration gradient tests).
References	[1]. Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry. Cytometry A. 2010 Nov; 77(11):1020-31.
Additional Infomation	Intracellular flow cytometry permits quantitation of diverse molecular targets at the single-cell level. However, limitations in detection sensitivity inherently restrict the method, sometimes resulting in the inability to measure proteins of very low abundance or to differentiate cells expressing subtly different protein concentrations. To improve these measurements, an enzymatic amplification approach called tyramide signal amplification (TSA) was optimized for assessment of intracellular kinase cascades. First, Pacific Blue, Pacific Orange, and Alexa Fluor 488 tyramide reporters were shown to exhibit low nonspecific binding in permeabilized cells. Next, the effects of antibody concentration, tyramide concentration, and reaction time on assay resolution were characterized. Use of optimized TSA resulted in a 10-fold or greater improvement in measurement resolution of endogenous Erk and Stat cell signaling pathways relative to standard, nonamplified detection. TSA also enhanced assay sensitivity and, in conjunction with fluorescent cell barcoding, improved assay performance according to a metric used to evaluate high-throughput drug screens. TSA was used to profile Stat1 phosphorylation in primary immune system cells, which revealed heterogeneity in various populations, including CD4+ FoxP3+ regulatory T cells. We anticipate the approach will be broadly applicable to intracellular flow cytometry assays with low signal-to-noise ratios.[1]

Solubility Data

Solubility (In Vitro)

DMSO :~100 mg/mL (~110.72 mM; with sonication (<60°C))

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.1072 mL	5.5359 mL	11.0717 mL
	5 mM	0.2214 mL	1.1072 mL	2.2143 mL
	10 mM	0.1107 mL	0.5536 mL	1.1072 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.