TO-PRO®-3 Stain Protocol
Nuclear stain for fixed cells
TO-PRO®-3 is a cell-impermeant dye that gives strong and selective staining of the nucleus in cultured cells and paraffin sections. TO-PRO®-3 and other carbocyanine monomers have very strong binding affinity for dsDNA, with dissociation constants in the micromolar range. The long-wavelength fluorescence of TO-PRO®-3 is outside the spectral range of tissue autofluorescence as well as commonly used fluorophores in the DAPI and FITC channels.
This protocol can be used for:
- Nucleic acid (nuclear) staining in fluorescence microscopy
This protocol should not be used for:
- Flow cytometry
You will need the following for this protocol:
- Cells growing in culture
- TO-PRO®-3 stain (Cat. No. T3605)
- Fluorescence microscope
Labeling fixed cells
First, fix and permeabilize cultured cells with a protocol appropriate for your sample.
1. Wash the cells 1–3 times in PBS as needed.
2. Prepare the TO-PRO®-3 staining solution by diluting the TO-PRO®-3 stock solution 1:1,000 (1 µM) in PBS.
|3. Add sufficient staining solution to cover the cells.|
|4. Incubate for 15–30 minutes, protected from light.|
|5. Remove the staining solution.|
|6. Wash the cells 3 times in PBS.|
|7. Image the cells.|
|Standard filter set||Far Red|
|EVOS® Light Cube||Cy®5|
- Warm to room temperature and briefly centrifuge the DMSO solution to the bottom of the vial each time before use.
- Try multiple dye concentrations in the range from 100 nM to 5 µM to determine the optimal concentration.
- RNase treatment is not necessary but could improve nuclear signals over cytoplasmic RNA background.
- Treat all nucleic acid binding dyes as potential mutagens and handle with care.