NRTP

Guidelines
(The recommended experimental protocol below is for reference only and needs to be adjusted according to your specific requirements)^[1].

1. Working Solution Preparation
1.2 Concentration recommendation: 5 μM.

2. Staining Procedures
2.1 Sample type: Adherent cells (Hepg2 cells)
2.1.1 Adherent cells: No trypsin digestion is required before imaging.
2.2 Incubation conditions: Incubate with 5 μM NRTP for 30 min.
2.3 Washing step: No washing is needed after incubation.

3. Control Setup
3.1 Use the mitochondria-targeted probe MTDR (HY-D1783) as a positive control for colocalization validation.
3.2 Set up a blank control containing only medium to eliminate background interference.

4. Detection and Analysis
4.1 Instrument: Confocal fluorescence microscope
4.1.1 Excitation wavelength: 561 nm; collect emission light in the red channel (570-620 nm) and deep red channel (665-735 nm), respectively.
4.2 Result analysis:
4.2.1 Fluorescence intensity changes: In a low-polarity environment, red fluorescence intensity increases while deep red fluorescence intensity decreases; in a high-polarity environment, both red and deep red fluorescence show high intensity.
4.2.2 Fluorescence localization: Mitochondria.
4.2.3 Color changes: The red channel shows red fluorescence, and the deep red channel shows deep red fluorescence; merged images exhibit yellow/orange (high polarity) or red (low polarity) pseudocolor; ratio images exhibit yellowish-green (high polarity) to cyan (low polarity) pseudocolor.