MAO probe 1 can be used as a substrate for purifying MAO-A and MAO-B. In HEPES buffer at pH 7.4, the Km values measured at 25°C were 70 μM (MAO-A) and 75 μM (MAO-B), the kcat values were 71 min⁻¹ (MAO-A) and 53 min⁻¹ (MAO-B), and the kobs values were 5.8 s⁻¹ (MAO-A) and 6.4 s⁻¹ (MAO-B) [1]. When MAO probe 1 (10 μM; 1 h) was incubated with aggregated Aβ1-42 plaques in PBS buffer (pH 7.4) at 25°C, no fluorescence response was observed [1].

MAO probe 1 (10 mg/kg; intraperitoneal injection; single dose) can be used to simultaneously monitor MAO activity and Aβ plaque burden in 5XFAD mice in vivo. Its fluorescence intensity (reflecting MAO activity and AD progression) shows three different stage-specific changes with age: a slow onset stage before 3 months of age, a rapid progression stage from 4 to 9 months of age, and a plateau stage after 9 months of age [1]. MAO probe 1 (10 mg/kg; intraperitoneal injection; single dose) showed only negligible fluorescence signal in 9-month-old healthy wild-type mice, confirming that it can distinguish between healthy mice and Alzheimer's disease mice [1]. Experimental Guide (The following is our recommended experimental protocol. This protocol is for reference only, and the specific operation should be adjusted according to actual needs.) 1. Preparation of probe 1/IBC 2 stock solution: Dissolve in DMSO. Prepare a high concentration stock solution (usually 10-20 mM) and store it in the dark at -20°C. Dilute the working solution with PBS/aCSF to the required concentration (10 μM for tissue staining; adjust the concentration according to body weight for in vivo experiments). 2. In vivo two-photon co-monitoring of MAO activity + Aβ plaques 2.1 Animal model: 2-11 month old 5XFAD transgenic mice (Tg6799) and age-matched wild-type control mice 2.2 Probe injection: Intraperitoneal injection of probe 1 at a dose of 10 mg/kg body weight. For example: 20 g mouse → 200 μg probe 1, dissolved in a suitable solvent (commonly 5% DMSO + PBS or physiological saline). After injection, incubate for 2 hours to allow the probe to cross the blood-brain barrier and be converted to IBC by MAO. 2.3 Surgical preparation (thin craniotomy/craniotomy) 2.3.1 Anesthesia: Zolectin 50 + Ropronin (1.2 mL/kg, intramuscular injection), fixed on a 37℃ heating plate. 2.3.2 Disinfect and harvest scalp, selecting an area 0.5-1 mm from the anterior fontanelle, and grind the skull to approximately 30 μm. 2.3.3 Fix with a round coverslip and dental adhesive. 2.4 Two-photon imaging excitation wavelength: 850 nm; emission and acquisition: 570-620 nm (red channel, IBC 2); dextran-Texas red (blood vessels, approximately 590-650 nm) can be acquired simultaneously. 2.5 Control group (optional): One week prior to the experiment, selegiline (MAO-B inhibitor, 10 mg/150 mL, freely drinkable) was added to drinking water, and the same procedure was performed. A significant signal reduction was expected. 3. Aβ plaque staining of ex vivo brain tissue sections 3.1 Bloodletting of 5XFAD mice and rapid removal of brain tissue. 3.2 Horizontal sections of the hippocampus were prepared using a vibratory microtome (Leica) (operated on ice, continuously perfused with artificial cerebrospinal fluid containing 95% O₂/5% CO₂). The sections were placed in artificial cerebrospinal fluid containing IBC 2 (10 μM) + MeO-XO4 (10 μM, Aβ reference dye) and incubated at 33°C for 45 minutes. 3.3 Wash 2-3 times with artificial cerebrospinal fluid. 3.4 Two-photon microscopy imaging: excitation wavelength 850 nm, emission wavelength 570-620 nm. Note: In vitro experiments can also use IBC 2 to directly stain plaques for verification; in vivo experiments only use probe 1 (IBC 2 generated in situ from MAO).

Animal/Disease Models:5XFAD Tg (Tg6799, 2-11 months old)[1]
Doses: 10 mg/kg
Route of Administration: Intraperitoneal injection; single dose
Experimental Results: The results showed that both background fluorescence (associated with MAO activity) and plaque-specific fluorescence (associated with Aβ plaque burden) increased significantly with age. Background fluorescence and plaque fluorescence in 2-3 month old mice were negligible, increased significantly from 4 months old, rose rapidly at 9-10 months old, and reached saturation at 11 months old. Cerebral amyloid angiopathy was detected in 4-month-old mice. In 9-month-old Alzheimer's disease mice pretreated with the monoamine oxidase inhibitor selegiline, both background fluorescence and plaque fluorescence signals were significantly reduced. Background fluorescence intensity (representing monoamine oxidase activity) was observed to be positively correlated with plaque volume per cubic millimeter of brain tissue.

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Animal/Disease Models:Wild-type littermates (9 months old) [1]
Doses: 10 mg/kg
Route of Administration: Intraperitoneal injection; single administration
Experimental Results: Negligible background and plaque-specific fluorescence signals were detected, indicating that MAO activity was extremely low and Aβ plaques were not present in healthy mice.

[1]. Close Correlation of Monoamine Oxidase Activity with Progress of Alzheimer's Disease in Mice, Observed by in Vivo Two-Photon Imaging. ACS Cent Sci. 2016;2(12):967-975.

C19H20N4O

320.39

1386860-79-2

Typically exists as solids at room temperature

NCCCOC1=CC2=CC(N(C)C)=CC=C2C=C1/C=C(C#N)/C#N

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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

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.)