Fluorescent Dye

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).

[1]. Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry. Cytometry A. 2010 Nov; 77(11):1020-31.

Intracellular flow cytometry enables the quantitative analysis of a wide range of molecular targets at the single-cell level. However, limitations in detection sensitivity restrict the application of this method, sometimes resulting in the inability to measure proteins with extremely low abundance or to distinguish cells expressing proteins at slightly different concentrations. To improve these measurements, we optimized an enzymatic amplification method called tyramine signal amplification (TSA) to assess intracellular kinase cascades. First, we demonstrated that Pacific Blue, Pacific Orange, and Alexa Fluor 488 tyramine reporter molecules have low nonspecific binding in permeabilized cells. Next, we investigated the effects of antibody concentration, tyramine concentration, and reaction time on detection resolution. Compared to standard non-amplified detection methods, the optimized TSA method improved the measurement resolution of endogenous Erk and Stat cell signaling pathways by 10-fold or more. TSA also improved detection sensitivity and, when combined with fluorescent cell barcoding, enhanced detection performance based on metrics used to assess high-throughput drug screening. TSA was used to analyze Stat1 phosphorylation in primary immune system cells, revealing heterogeneity among different cell populations, including CD4+FoxP3+ regulatory T cells. We expect this method to be widely applicable to intracellular flow cytometry analysis with low signal-to-noise ratios. [1]

C49H66N4O8S2

903.20

902.432207

169494300

Dark green to black solid powder

3

9

17

63

1740

0

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

PKVPZSGTNHZHDH-UHFFFAOYSA-N

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

(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

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

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.

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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).

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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)

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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).

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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

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)