Azure B (Azure B chloride) is a cationic dye and the major metabolite of Methylene blue. Azure B is a selective, and reversible inhibitor of MAO-A (IC50s: 11 and 968 nM for recombinant human MAO-A and MAO-B).

MAO-B:968 nM, MAO-A:11 nM

Azure B (4-30 mg/kg; i.p.; once) reduces immobility in the forced swim test [1].

Instructions
1. Solution configuration
1. Configuration of mother liquor: Use sterile water or PBS to configure MHI-148 into 1-10mM mother liquor and store it at -20℃ or -80℃. Please aliquot it to avoid repeated freeze-thawing.
2. Configuration of working fluid: When in use, dilute the MHI-148 mother liquor into a working fluid that meets the requirements and adjust according to the experimental requirements.
2. Blood smear staining:
1. Prepare an Azure B staining solution, usually by dissolving Azure B chloride in buffer or solvent.
2. Apply the staining solution to the prepared blood smear.
3. Allow the smear to incubate in solution for a few minutes to promote cell staining.
4. Rinse the smear thoroughly with water to remove excess dye and then air dry.
5. Check the stained smear under a microscope and analyze the morphology of blood cells.
3. Monoamine oxidase (MAO) inhibition:
1. Introduce Azure B into in vitro experiments, the inhibitory effect is usually determined using recombinant human MAO-A and MAO-B.
2. The IC50 value at a specific experimental setting can be determined through dose response studies.
3. In addition, the antidepressant effect of Azure B can be evaluated in animal models through behavioral experiments (such as forced swimming experiments, tail suspension experiments).