- 1. Prepare mitochondrial staining solution
a. Reconstitute 0.5 mg IraZolve-Mito with 55 μL DMSO to prepare a 10 mM IraZolve-Mito stock solution.
b. Store IraZolve-Mito stock solution at 4°C protected from light.
Note 1: IraZolve-Mito stock solution should be used within two months of reconstitution for best staining results.
- 2. Prepare and stain live cells
a. Adherent cells:
i．Grow cells in 6-well plate on coverslips to desired confluency (~70-80%).
ii. Remove culture medium and add pre-warmed PBS (37°C) or serum-free medium containing IraZolve-Mito at a final concentration of 10-50 μM.
iii. Incubate cells at 37°C, 5% CO2 for 30 minutes.
iv. Wash cells 2 x 1 minute in PBS.
v. Mount coverslips in aqueous mounting media.
vi. Observe cells using fluorescence technique of choice.
Note 2: Glycerol based mounting media may reduce fluorescence intensity of IraZolve-Mitomm
b. Suspension cells:
i．Pellet cell suspension and remove supernatant.
ii. Resuspend cells in pre-warmed PBS (37°C) or serum-free medium containing IraZolve-Mito at a final concentration of 10-50 μM.
iii. Incubate cells at 37°C, 5% CO2 for 30 minutes.
iv. Re-pellet the cells and resuspend in PBS or serum-free medium.
v. Pipette cells onto a coverslip for imaging in PBS or serum-free medium OR adhere cells to a poly-L-lysine (or similar) coated coverslip by pipetting cells onto the coverslip, allow cells to settle for 2-5 minutes, and wet mount coverslip.
vi. Observe using fluorescence technique of choice.
Note 3: Optimal staining may vary between cell lines. Staining conditions may be modified according to cell type.
Note 4: For epifluorescence applications, IraZolve-Mito can be excited at approximately 365 nm (UV) or405 nm. For confocal and two-photon applications, it can be excited at 400 nm and 800-830 nm, respectively.
- 3. Prepare and stain tissue samples**
**Note 5: Tissue can be stained immediately upon collection or stored for later staining. IraZolve-Mito is compatible with tissue preserved using 4% paraformaldehyde fixation and flash freezing.
Note 6: To quench endogenous fluorescence, incubate samples in PBS (pH 7.4) with 100 mM glycine for 20 minutes at room temperature. Other methods to quench fluorescence may be used, such as UV irradiation, however, harsh treatments may induce lipid leaching and/or interfere with lipid binding and should be avoided.
a. Staining tissue sections:
i. Incubate fresh, fixed, or thawed tissue samples with PBS containing IraZolve-Mito at a final concentration of 10-50μM for 30 minutes at room temperature.
ii. Wash samples 3 x 5 minutes in PBS.
iii. Mount coverslips using aqueous mounting media.
iv. Observe using fluorescence technique of choice.
This protocol only provides a guideline, and should be modified according to your specific needs.
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