| Size | Price | Stock | Qty |
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| 1mg |
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| Other Sizes |
| Targets |
True Blue targets neurons for retrograde tracing, specifically labeling the nucleus, nucleolus, cell body, proximal dendrites, and axons. The dye is taken up by axon terminals and transported retrogradely to the cell body and dendrites, providing detailed visualization of the entire neuronal morphology. It is a UV light-excitable, divalent cationic dye that stains the cytoplasm with blue fluorescence. Unlike some other retrograde tracers, True Blue has no effects on neuronal survival, whether studied 4 days or 20 weeks after application. This property makes it safe for long-term tracing studies. True Blue can label dorsal root ganglion neurons (DRGNs) and motoneurons (MNs).
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| ln Vitro |
In vitro, True Blue is used for fluorescence-based neuronal tracing applications. The dye is applied to neuronal cell cultures or to the cut ends of nerves in explant cultures, where it is taken up by axon terminals and transported retrogradely to the cell bodies. The excitation/emission maxima are 373/404 nm (or 395-425 nm excitation with a 450 nm barrier filter for standard filter sets), and the blue fluorescence allows for clear visualization of neuronal morphology. The staining pattern is distinct, with the nucleus and nucleolus prominently labeled in addition to the cytoplasm, axons, and dendrites. The dye shows stability in immunohistochemical processing, enabling combination with other fluorescent labels.
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| ln Vivo |
Adult female rats' severed sciatic nerves can be submerged in True Blue to label dorsal root ganglion neurons (DRGN) and motor neurons (MN) without compromising neuronal survival four days or twenty weeks later [2].
In vivo, True Blue is used as a retrograde tracer in animal models (primarily rats and mice) to map neuronal connections and pathways. The dye is applied to the cut end of a nerve (e.g., sciatic nerve) or injected into a specific brain region. The dye is taken up by axon terminals and transported retrogradely to the cell body and dendrites of neurons projecting to that area. After a survival period of 3-14 days (for optimal transport), animals are euthanized, and the brain or spinal cord is sectioned and examined under a fluorescence microscope (UV excitation). True Blue has no effect on neuronal survival, allowing for long-term tracing studies over weeks to months. It can label dorsal root ganglion neurons and motoneurons. |
| Enzyme Assay |
For non-cell-based assays, True Blue is characterized by UV-Vis absorption and fluorescence spectroscopy. A stock solution of True Blue (1-5 mg/mL) is prepared in distilled water, PBS, or 10% DMSO. For absorption measurement, the solution is diluted to 10-50 ug/mL in PBS (pH 7.4), and the absorption spectrum is recorded from 250-500 nm. True Blue has absorption maxima at approximately 373 nm and a shoulder at 395-425 nm. For fluorescence measurement, the solution is excited at 373 nm, and the emission spectrum is recorded from 380-600 nm, with the emission maximum near 404 nm. For neuronal labeling experiments, the dye is applied to the cut ends of nerves or injected into brain regions as described.
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| Cell Assay |
For in vitro cell-based assays, primary neuronal cultures are prepared from embryonic or postnatal rodent brains. Neurons are seeded on coverslips and cultured for 7-14 days. True Blue (10-100 ug/mL) is added to the culture medium for 2-24 hours, or it is applied to the axons using compartmentalized culture systems. Cells are then washed, fixed with 4% paraformaldehyde, and mounted on slides. Fluorescence microscopy with UV excitation (filter set: 365-400 nm excitation, 420-450 nm emission) reveals the blue fluorescence in the neuronal cell bodies, dendrites, and axons. The dye is compatible with immunostaining for neuronal markers (e.g., NeuN, MAP2, neurofilament), and the blue emission does not significantly overlap with green or red fluorophores, enabling multicolor labeling.
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| Animal Protocol |
For in vivo animal studies, a standard protocol for retrograde tracing in the rat sciatic nerve is used. Adult female Sprague-Dawley rats (200-250 g) are anesthetized with isoflurane. The sciatic nerve is exposed, and a cut is made at the mid-thigh level. The proximal cut end is soaked in a solution of True Blue (2-10% w/v in distilled water or PBS) for 10-30 minutes. For injections into brain regions (e.g., striatum, cortex, hippocampus), 0.5-2 uL of 2-5% True Blue solution is stereotaxically injected at coordinates specific to the target region. After injection, the incision is closed, and the animal is allowed to recover. After a survival period of 3-14 days (to allow retrograde transport), the animal is euthanized, and the brain or spinal cord is harvested. The tissue is fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, sectioned (20-40 um thick) on a cryostat, and mounted on slides. Sections are examined under a fluorescence microscope with UV excitation. True Blue labels the nucleus, nucleolus, cell body, proximal dendrites, and axons of neurons projecting to the injection site.
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| ADME/Pharmacokinetics |
True Blue has a molecular weight of 417.3 g/mol and a molecular formula of C20H18Cl2N4O2. The compound is supplied as a light yellow to yellow solid powder with a purity of ≥90% (usually ≥96.8%). It is soluble in water, PBS, and DMSO. Stock solutions (1-5 mg/mL) are prepared in distilled water or PBS (pH 7.4). True Blue is a divalent cationic dye that is taken up by axon terminals via endocytosis and is transported retrogradely via the dynein motor protein system. The compound is stable in immunohistochemical processing, including fixation, permeabilization, and antibody staining. Storage conditions: 4degC or -20degC, protected from light. For long-term storage, store as a powder at -20degC.
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| Toxicity/Toxicokinetics |
True Blue is a fluorescent tracer and is not intended for human use. No significant toxicity has been reported at the concentrations used for neuronal tracing (0.5-10% solutions). In animal studies, the dye has no effect on neuronal survival, even after 20 weeks. Standard chemical safety precautions (gloves, lab coat, safety glasses) should be followed when handling the powder. Avoid inhalation of dust. The dye is not considered a carcinogen or reproductive hazard. Acute toxicity: The LD50 is not established, but the compound is expected to have low systemic toxicity due to the small volumes typically used for neuronal tracing (0.5-10% solutions, 0.5-2 uL for intracranial injections).
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| References |
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| Additional Infomation |
True Blue is a research compound and is not approved for clinical use. It is one of several fluorescent retrograde tracers commonly used in neuroscience research, alongside Fluorogold, DiI, and Fast Blue. True Blue has the advantage of labeling the nucleus and nucleolus prominently in addition to the cytoplasm, providing better visualization of neuronal cell bodies. The dye is UV-excitable and emits blue fluorescence, allowing for combination with other fluorophores for double- or triple-labeling studies. True Blue shows no cytotoxic effects on neurons, as demonstrated by studies showing no reduction in neuronal survival 4 days or 20 weeks after application. True Blue can label dorsal root ganglion neurons and motoneurons by soaking the cut sciatic nerve. The product is for research use only and is not for human therapeutic or diagnostic applications.
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| Molecular Formula |
C20H16N4O2.2[HCL]
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| Molecular Weight |
417.28852
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| Exact Mass |
416.081
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| CAS # |
71431-30-6
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| Related CAS # |
71431-30-6 (HCl);50638-24-9;
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| PubChem CID |
5706759
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| Appearance |
Light yellow to yellow solid powder
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| Boiling Point |
600.6ºC at 760 mmHg
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| Flash Point |
317.1ºC
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| LogP |
7.121
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| Hydrogen Bond Donor Count |
6
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
28
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| Complexity |
542
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC2=C(C=C1C(=N)N)C=C(O2)/C=C/C3=CC4=C(O3)C=CC(=C4)C(=N)N.Cl.Cl
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| InChi Key |
WFZFMHDDZRBTFH-CZEFNJPISA-N
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| InChi Code |
InChI=1S/C20H16N4O2.2ClH/c21-19(22)11-1-5-17-13(7-11)9-15(25-17)3-4-16-10-14-8-12(20(23)24)2-6-18(14)26-16;;/h1-10H,(H3,21,22)(H3,23,24);2*1H/b4-3+;;
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| Chemical Name |
2-[(E)-2-(5-carbamimidoyl-1-benzofuran-2-yl)ethenyl]-1-benzofuran-5-carboximidamide;dihydrochloride
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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 |
| 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 | 2.3964 mL | 11.9821 mL | 23.9641 mL | |
| 5 mM | 0.4793 mL | 2.3964 mL | 4.7928 mL | |
| 10 mM | 0.2396 mL | 1.1982 mL | 2.3964 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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