The optimal stock solution preparation 1. Protein preparation: Add protein (antibody) at a concentration of 2 mg/mL to achieve the labeling effect. 1) The protein solution's pH should be 8.5±0.5. In case the pH falls below 8.0, utilize 1 M carbon dioxide. 2) The labeling efficiency will be significantly decreased if the protein content is less than 2 mg/mL. The ideal labeling efficiency can be achieved by ensuring that the protein content ranges from 2 to 10 mg/mL. 3) To ensure optimal labeling efficacy, the protein needs to be in a clear buffer that contains primary amines (such Tris or glycine) and ammonium ions. 2. Dye preparation: To generate a 10 mM stock solution, add anhydrous DMSO to CY dye. Using a glass tube or a vortex, thoroughly mix. After being aliquoted, it is advised to store CY storage solution at -20°C or -80°C in the dark. 3. Amount of dye working solution The amount of labeled protein determines how much CY dye is needed for the labeling reaction. The ideal amount of CY dye and protein to utilize is as follows: Assume 500 μL of labeled IgG (2 mg/mL; MW = 150,000). Dissolve a tube of 1 mg CY dye in 100 μL of DMSO. This means that 3.95 μL of CY volume is needed. Using CY3-NHS ester as an example, the calculation procedure is as follows in detail: 1) mmol (IgG) = mg/mL (IgG) ×mL (IgG) / MW (IgG) = 2 mg/mL×0.5 mL / 150,000 mg/mmol = 6.7×10-6 mmol 2) mmol (CY3-NHS ester) = mmol (IgG) ×10 = 6.7×10-6 mmol×10 = 6.7×10-5 mmol 3) μL (CY3-NHS ester) = mmol (CY3-NHS ester) ×MW (CY3-NHS ester) / mg/μL (CY3-NHS ester) = 6.7×10-5 mmol× 590.15 mg/mmol / 0.01 mg/μL = 3.95 μL (CY3-NHS ester) Usage method 1. Labeling reaction 1: Add 0.5 mL of the protein sample solution to the predicted amount of a fresh carrier containing 10 mg/mL CY dye. Gently shake to combine, and then remove the centrifuged material by briefly collecting it at the bottom of the reaction tube. Avoid copying 2) Place the reaction tube in a dark location, give it a little shake, and walk for 60 minutes under the initial conditions. Every week, for ten to fifteen minutes, carefully flip the reaction tube over many times to 2. Protein blocking and desalting As an example, the SepHadex G-25 column blocked dye conjugates are used in the methodology that follows. 1) As directed by the manufacturer, set up the SepHadex G-25 column. 2) Place the reaction mixture into the SepHadex G-25 column's upper section. 3) Add PBS (pH 7.2–7.4) when the sample dips below the top resin's surface. 4) Immediately add extra PBS (pH 7.2-7.4) to the needed sample to finish columnar shrinking. Put the parts together that contain the chosen dye-protein combination. Notes: 1. CY dye is sensitive to light and humidity. Prepare CY solution immediately and discard unused part. 2. Low amounts of sodium azide (≤3 mM or 0.02%) or thimerosal (≤0.02 mM or 0.01%) will not significantly impair protein labeling; while 20-50% glycerol will reduce labeling efficiency. 3. Avoid utilizing buffers containing primary amines (such as Tris, glycine) or ammonium ions. They will compete with the protein to be tagged and impair labeling efficiency. 4. For unactivated dyes, condensation solution (500 ug/mL) needs to be used first (25952-53-8 5. This product is only for scientific research by professionals and must not be used for clinical diagnosis or treatment, and must not be used in food or Medications. 6. For your safety and health, please wear a lab coat and keep an eye on the operation.

[1]. Cyanine fluorophore derivatives with enhanced photostability. Nat Methods. 2011 Nov 13;9(1):68-71.

[2]. Shindy, H. A. (2017). Fundamentals in the chemistry of cyanine dyes: A review. Dyes and Pigments, 145, 505–513. doi:10.1016/j.dyepig.2017.06.029.

C25H26N2O4

418.484946727753

418.189

260430-02-2

Cy2 (iodine)

9953812

Pink to red solid powder

6

0

5

8

31

658

0

CCN\1C2=CC=CC=C2O/C1=C/C=C/C3=[N+](C4=CC=CC=C4O3)CCCCCC(=O)[O-]

WORLWSFCGZCFSW-UHFFFAOYSA-N

InChI=1S/C25H26N2O4/c1-2-26-19-11-5-7-13-21(19)30-23(26)15-10-16-24-27(18-9-3-4-17-25(28)29)20-12-6-8-14-22(20)31-24/h5-8,10-16H,2-4,9,17-18H2,1H3

6-[2-[(E,3E)-3-(3-ethyl-1,3-benzoxazol-2-ylidene)prop-1-enyl]-1,3-benzoxazol-3-ium-3-yl]hexanoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ≥ 30 mg/mL (~71.69 mM)

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.)