ALDH2 probe 5

Operating Instructions
(The following is a recommended experimental protocol for guidance only; please adjust it according to your own experimental requirements)

1. Stock Solution Preparation
1.1 Solvent: Most dyes are typically dissolved in organic solvents, such as anhydrous DMSO.
1.1 Concentration Recommendation: It is generally recommended to prepare a high-concentration stock solution of 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: 10 mM PBS, pH 7.4.
2.2 Working Concentration: 1-10 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedure
3.1 Sample Types^[1]:
3.1.1 Live cells (BV2 cells).
3.1.2 Blood samples.
3.1.3 Intact mouse brain tissues.
3.1.4 Tissue homogenates.
3.2 Incubation Conditions: Co-incubate with NAD+ (HY-B0445) (2.5 mM) for 2 h, 10 min, or up to 30 min; use working concentrations of 1 μM, 8 μM, or 10 μM.

4. Control Setup
4.1 Set up negative controls, including samples without ALDH2, blank controls, and controls containing other ALDH isoforms or non-target proteins.
4.2 Positive controls are used to validate the experimental system; blank controls are used to exclude fluorescent interference from reagents.

5. Detection and Analysis
5.1 Instrument: Fluorescence spectrometer.
5.1.1 Excitation/Emission Wavelength: The maximum emission wavelength is approximately 494 nm to 500 nm.
5.2 Result Analysis:
5.2.2 Fluorescence intensity increases by >220-fold after ALDH2-mediated conversion of probe A5; fluorescence intensity correlates with probe concentration and ALDH2 concentration; the fluorescence intensity of tissue homogenates increases with prolonged incubation time.
5.2.2 Fluorescence localizes to mitochondria (targeting mitochondrial ALDH2).
5.2.3 Probes without fluorescent activity emit bright fluorescence after catalytic oxidation by ALDH2.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.