| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| 5mg |
|
||
| Other Sizes |
| Targets |
Cyanine 3 Tyramide methyl indole is an enzyme substrate, not a receptor ligand. The target is horseradish peroxidase (HRP), a commonly used detection enzyme conjugated to secondary antibodies or streptavidin. In TSA, HRP catalyzes the conversion of the tyramide to a highly reactive radical, which then forms covalent bonds with electron-rich aromatic amino acids (tyrosine, tryptophan) adjacent to the HRP-probe binding site. This deposition amplifies the signal associated with the primary antigen or nucleic acid target.
|
|---|---|
| ln Vitro |
The methyl indole derivative of Cy3 Tyramide is chemically optimized for brighter fluorescence and improved stability in aqueous amplification buffers compared to standard TSA reagents. In standard fluorescent TSA assays, the Cy3 tyramide provides a high signal-to-noise ratio with minimal non-specific background. The orange fluorescence (Ex ~550 nm, Em ~570 nm) is compatible with common green (FITC, 488 nm excitation) and far-red (Cy5, 640 nm) fluorophores, enabling multi-color detection. No direct pharmacological effects are observed.
|
| ln Vivo |
There is no in vivo activity, as the compound is strictly used for ex vivo staining of fixed biological specimens (formalin-fixed, paraffin-embedded (FFPE) tissue sections, frozen sections, cell smears, and cultured cells on slides). However, this TSA method is extensively applied in preclinical studies to quantify biomarkers in tissues collected from drug-treated animals (e.g., xenograft mouse models). The increased sensitivity (up to 100-fold over standard IHC) allows for reliable detection of low-abundance phosphorylated proteins or transcription factors.
|
| Enzyme Assay |
The HRP-catalyzed deposition is assessed in a cell-free system. A 96-well microplate is coated with BSA (10 microg/well). HRP (0.1-10 ng/well) in PBS is added and incubated for 1 hour, followed by blocking with 1% BSA. Cyanine 3 Tyramide methyl indole (diluted 1:100 from a stock solution, typically 10 microM final concentration) is added with H2O2 (0.01% final) in TSA amplification buffer (0.1 M borate buffer, pH 8.5, containing 0.1% Tween-20 and 10% dextran sulfate). After 10-30 minutes, the reaction is stopped with 0.1% sodium azide. Plates are washed, and fluorescence is measured (Ex 530-550 nm, Em 565-580 nm). A linear relationship between HRP concentration and fluorescence signal (R2 > 0.99) confirms proper enzyme kinetics. Specificity controls include omission of H2O2 (no signal) or omission of HRP (no signal).
|
| Cell Assay |
Cyanine 3 Tyramide methyl indole is used for immunofluorescence staining on cells grown on coverslips, cytospins, or chamber slides. A standard protocol: 1) Fix cells with 4% paraformaldehyde for 15 minutes. 2) Permeabilize with 0.1-0.5% Triton X-100 in PBS for 10 minutes. 3) Quench endogenous peroxidase activity with 3% H2O2 in PBS for 10 minutes. 4) Block with 5% normal serum (same species as secondary antibody) in PBS for 1 hour. 5) Incubate with primary antibody (e.g., anti-active caspase-3, anti-Phospho-ERK, etc.) diluted in blocking buffer overnight at 4degC. 6) Incubate with HRP-conjugated secondary antibody (1:200-1:500) for 30 minutes at room temperature. 7) Apply Cyanine 3 Tyramide working solution (diluted 1:50 to 1:200 in the manufacturer's amplification buffer containing freshly added 0.01% H2O2) for 5-15 minutes. 8) Wash 3×5 minutes with TBS-Tween. 9) Counterstain nuclei with DAPI (1 microg/mL) for 5 minutes. 10) Mount with anti-fade medium and image by fluorescence microscopy (Ex 550 nm, Em 570 nm). The staining pattern should be punctate, nuclear, or cytoplasmic depending on the antigen. A signal amplification step can be repeated for multiplexing.
|
| Animal Protocol |
Since this is a staining reagent, it is not administered to animals. However, the performance is often validated on tissue sections from animal disease models. For example, in a mouse xenograft model treated with an anti-cancer drug, tumor tissues are harvested, fixed in 10% formalin, embedded in paraffin, and sectioned (4 microm). The sections are processed as described in the “In Vitro Cell Experiment Protocol” section. After staining with Cy3 tyramide, slides are scanned using a whole-slide fluorescence scanner. The staining intensity is quantified by image analysis software (e.g., HALO, QuPath, Visiopharm) to compute an H-score or percentage of positive cells. A higher signal-to-noise ratio compared to direct immunofluorescence is expected.
|
| ADME/Pharmacokinetics |
The compound is used as a bench-top staining reagent. No pharmacokinetic studies are performed. When stored at -20degC as a powder or concentrated DMSO stock solution, Cyanine 3 Tyramide methyl indole is stable for at least 12 months. Protect from light at all times. The working solution in aqueous buffer should be prepared immediately before use and discarded after the experiment because H2O2 induces hydrolysis of the tyramide over time. The reaction product (covalently bound dye to tissue) is stable for months when stored in the dark at 4degC.
|
| Toxicity/Toxicokinetics |
As a research chemical, Cyanine 3 Tyramide methyl indole is not classified as highly toxic. Standard safe handling practices: avoid contact with skin and eyes; wash hands after handling. The compound may be irritating to the respiratory tract if inhaled as a powder. Acute toxicity tests are not available, but the LD50 in rodents is expected to be >500 mg/kg. The tyramide is not known to be genotoxic. As with all fluorescent dyes, avoid direct ingestion and inhalation. Dispose of staining solutions (contains H2O2 and organic dye) according to biohazardous waste (if tissues used) or hazardous chemical waste protocols. The reagent is for research use only.
|
| References |
|
| Additional Infomation |
Cyanine 3 Tyramide methyl indole is an orange-emitting (Cy3, ~550/570 nm) tyramide derivative used in tyramide signal amplification (TSA). TSA is a powerful method for detecting low-abundance targets (down to a few copies per cell). The methyl indole moiety likely refers to a structural modification of the Cy3 fluorophore that enhances photostability and binding efficiency. Compared to direct fluorescent secondary antibodies, TSA increases signal strength by 10-100 fold, enabling the use of less primary antibody, which reduces background and conserves precious antibodies. Cy3 is a popular choice for two-color experiments (with Cy5 for a second channel) because its emission does not overlap with green (FITC) or far-red (Cy5) dyes. The compound is for research use only and not for human diagnostic or therapeutic applications.
|
| Molecular Formula |
C38H45N3O8S2
|
|---|---|
| Molecular Weight |
735.91
|
| Related CAS # |
Cyanine 3 Tyramide;174961-75-2
|
| Appearance |
Brown to dark brown solid powder
|
| 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: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). 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)
|
| 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
|
|---|---|
| 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 | 1.3589 mL | 6.7943 mL | 13.5886 mL | |
| 5 mM | 0.2718 mL | 1.3589 mL | 2.7177 mL | |
| 10 mM | 0.1359 mL | 0.6794 mL | 1.3589 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
