MBCB

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: No specific solvent stated.
1.2 Concentration recommendation: 10 μM

2. Working Solution Preparation
2.1 Diluent: PBS (20 mM, pH 7.4, containing 20% glycerol)
2.2 Working concentration: 10 μM
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type:[1]
3.1.1 Adherent cells: HeLa cells (no trypsinization step specified for imaging)
3.1.2 In vivo sample: 5-day-old zebrafishes
3.2 Incubation conditions:
3.2.1 For HeLa cells (SO₂ derivatives detection): Incubate with 10 μM MBCB for 0.5 h; optionally incubate with 10 μM HSO₃^- for another 0.5 h.
3.2.2 For HeLa cells (viscosity detection): Incubate with 10 μM MBCB for 0.5 h at 25 °C or 37 °C; alternatively, pre-incubate cells with 10 μM polyene antifungal agent for 1 h, then incubate with 10 μM MBCB for 0.5 h.
3.2.3 For zebrafishes: Incubate with 10 μM MBCB for 0.5 h; optionally incubate with 100 μM HSO₃^- for another 0.5 h.
3.3 Washing steps: No specific washing steps stated.

4. Controls
4.1 Set up negative control (cells/zebrafishes incubated with MBCB only)
4.2 Set up positive control (cells/zebrafishes incubated with MBCB plus HSO₃^-; cells incubated with MBCB plus polyene antifungal agent)

5. Detection & Analysis
5.1 Instrument: Two-photon confocal fluorescence microscope
5.1.1 For SO₂ derivatives detection: Ex = 740 nm; Em = 420-460 nm (blue channel) and 570-610 nm (red channel)
5.1.2 For viscosity detection: Ex = 740 nm; Em = 420-460 nm (blue channel) and 570-610 nm (red channel)
5.2 Result analysis:
5.2.1 Fluorescence intensity changes:
5.2.1.1 Upon interaction with HSO₃^-: Blue channel fluorescence intensity increases, while red channel fluorescence intensity decreases.
5.2.1.2 With increased viscosity (lower temperature or polyene antifungal agent treatment): Red channel fluorescence intensity increases, while blue channel fluorescence intensity remains nearly unchanged.
5.2.2 Fluorescence localization: Mitochondria in HeLa cells (Pearson's colocalization coefficient = 0.93 with Mito-Tracker Green); concentrated in zebrafish liver after HSO₃^- treatment.
5.2.3 Color changes: From red to blue upon HSO₃^- treatment; red fluorescence intensity increases with elevated mitochondrial viscosity