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| Targets |
The primary target for enzymatic activation is horseradish peroxidase (HRP), which is an enzyme often conjugated to a primary or secondary antibody, or to a streptavidin complex in a detection system. In the presence of hydrogen peroxide (H2O2), HRP catalyzes the oxidation of the tyramide moiety, generating a tyramide radical that covalently attaches to tyrosine residues in the vicinity of the HRP-labeled probe. There is no specific receptor target; the deposition occurs in a proximity-dependent manner (within 5-10 nm of the HRP molecule).
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| ln Vitro |
Cyanine 5 Tyramide itself has no intrinsic in vitro pharmacological activity; its utility lies in enzymatic signal amplification. In TSA systems, the dye provides a 10-100 fold increase in fluorescence signal intensity compared to direct fluorescent labeling methods. When applied to tissue sections or cell smears, the signal-to-noise ratio is dramatically improved, enabling the detection of low-abundance targets (e.g., rare mRNA transcripts, weakly expressed proteins). The red emission (Cy5) enables multiplexing with green (FITC) and far-red (Cy7) fluorophores.
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| ln Vivo |
In vivo activity is not applicable because TSA is a technique for ex vivo tissue staining (fixed tissue sections, cytospins, or cultured cell monolayers). The HRP reaction is performed on fixed, permeabilized samples, not on live animals. However, the technology has been validated in hundreds of ex vivo studies for biomarker detection in mouse, rat, and human tissues from xenograft tumor models, transgenic mouse models, and disease models. The enhanced sensitivity helps quantify low-level protein expression.
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| Enzyme Assay |
The tyramide deposition mechanism is not studied in cell-free receptor binding assays because it involves an enzymatic reaction. However, the HRP-catalyzed activation kinetics can be characterized in a cell-free system using immobilized BSA (bovine serum albumin). Briefly, HRP (0.1-1 microg/mL) is adsorbed onto the wells of a 96-well microtiter plate or onto a nitrocellulose membrane. Cyanine 5 Tyramide (1-10 microM) and H2O2 (0.01-0.1%) are added in Tris-buffered saline (TBS, pH 7.4). After 5-15 minutes, the reaction is stopped by adding 0.1% sodium azide. The wells or membrane spots are washed with TBS-Tween. The amount of deposited dye is quantified by fluorescence plate reading or densitometry. Signal intensity is directly proportional to HRP concentration and tyramide concentration.
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| Cell Assay |
Cyanine 5 Tyramide methyl indole is used for immunohistochemistry (IHC) or immunofluorescence (IF) on fixed cells or tissue sections, and for in situ hybridization (ISH) (detection of mRNA or miRNA). A typical IHC protocol: 1) Tissue sections (4 microm thick) are deparaffinized, rehydrated, and subjected to antigen retrieval (heat-induced epitope retrieval using citrate buffer, pH 6.0, or Tris-EDTA, pH 9.0). 2) Endogenous peroxidase activity is blocked with 3% H2O2 in PBS for 10-15 minutes. 3) Sections are blocked with 5% normal serum or BSA for 1 hour. 4) Primary antibody (against target protein, e.g., Ki-67, HER2, p53) is applied overnight at 4degC. 5) HRP-conjugated secondary antibody is applied for 30-60 minutes at room temperature. 6) Cyanine 5 Tyramide working solution (prepared by diluting stock solution 1:50-1:200 in amplification buffer provided in a TSA kit) is applied for 5-15 minutes. 7) The reaction is stopped by washing with TBS-Tween. 8) Nuclei are counterstained with DAPI (blue). 9) Slides are mounted with anti-fade mounting medium and imaged by fluorescence microscopy (Ex 620-650 nm, Em 660-710 nm). This method yields punctate or diffuse red fluorescence specifically at the site of the target antigen.
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| Animal Protocol |
Cyanine 5 Tyramide is used for ex vivo tissue staining only, not for in vivo animal experiments. However, the technique is often applied to tissues derived from animal studies (e.g., xenograft tumors from mice treated with a test compound). The tissue handling protocol: After euthanasia, organs are fixed in 10% neutral buffered formalin for 24-48 hours, processed to paraffin blocks, sectioned, and stained as described under “In Vitro Cell Experiment Protocol.” There is no direct dosing of animals with the tyramide reagent. The outcome is qualitative or semi-quantitative analysis of target protein expression levels using fluorescence intensity (mean fluorescence intensity, MFI) or percent positive cells. Images are analyzed using ImageJ or commercial software.
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| ADME/Pharmacokinetics |
PK analysis is not applicable because the compound is not administered to living organisms. It is used exclusively as a histological staining reagent on fixed, dead tissue. In solution, the tyramide is stable at 4degC for several months. The working solution (prepared in amplification buffer) is unstable after mixing with H2O2 and should be used within minutes. To minimize background autofluorescence, the red-emitting Cy5 tyramide is preferred because biological tissues have low autofluorescence in the far-red region.
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| Toxicity/Toxicokinetics |
The compound is a solid or solution that is handled in a laboratory fume hood. The material may be irritating to eyes and skin. Acute toxicity data are not available but, as with most organic dyes, it is considered moderately toxic to aquatic organisms. For safe handling: wear nitrile gloves, a lab coat, and safety glasses. Avoid generating aerosols. In case of skin contact, wash with soap and copious water. The compound contains a reactive tyramide group that could potentially act as a hapten if injected, but it is not designed for systemic administration. Dispose in accordance with local regulations for hazardous chemical waste.
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| References | |
| Additional Infomation |
Cyanine 5 Tyramide methyl indole is part of the tyramide signal amplification (TSA) family, also known as catalyzed reporter deposition (CARD). TSA is one of the most sensitive signal amplification methods available, enabling detection of targets present at 1-10 copies per cell, with detection limits down to attomole levels. The methyl indole modification may improve the chemical stability and brightness of the fluorophore. Cy5 is a far-red dye (Ex/Em: ~650/670 nm) that avoids the green autofluorescence of red blood cells and lipofuscin. This product is for research use only and is not intended for diagnostic procedures in humans. Multiple manufacturers offer TSA Kits that include the tyramide reagent, HRP blocking reagent, and amplification buffers. Signal amplification is irreversible, meaning the dye is permanently covalently attached to the tissue, allowing for archival storage of slides. For multiplexing (detection of 2-4 targets), sequential TSA cycles can be performed (first target: Cy3 tyramide; second target: Cy5 tyramide; third target: Cy7 tyramide), with an antibody stripping step (using heat or H2O2) in between if needed.
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| Molecular Formula |
C40H47N3O8S2
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| Molecular Weight |
761.95
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| Related CAS # |
Cyanine 5 Tyramide;1431148-26-3
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| Appearance |
Purple to purplish red solid powder
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| HS Tariff Code |
2934.99.9001
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| 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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3124 mL | 6.5621 mL | 13.1242 mL | |
| 5 mM | 0.2625 mL | 1.3124 mL | 2.6248 mL | |
| 10 mM | 0.1312 mL | 0.6562 mL | 1.3124 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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