image of Molecular Probes® DAPI packaging

Nuclear counterstain for fluorescence microscopy

A popular nuclear and chromosome counterstain, DAPI (4′,6-diamidino-2-phenylindole) emits blue fluorescence upon binding to AT regions of DNA.

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:


Preparing solutions

1. Add 2 mL of deionized water (diH2O) or dimethylformamide (DMF) to the entire contents of the DAPI vial to make a 14.3 mM (5 mg/mL) DAPI stock solution. Note: DAPI has poor solubility in water, so sonicate as necessary to dissolve. The 5 mg/mL DAPI stock solution may be stored at 2–6°C for up to 6 months or at ≤–20°C for longer periods.
2. Add 2.1 µL of the 14.3 mM DAPI stock solution to 100 µL PBS to make a 300 µM DAPI intermediate dilution
3. Dilute the 300 µM DAPI intermediate dilution 1:1,000 in PBS as needed to make a 300 nM DAPI stain solution.

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. Add sufficient 300 nM DAPI stain solution to cover the cells.
3. Incubate for 1–5 minutes, protected from light.
4. Remove the stain solution.
5. Wash the cells 2–3 times in PBS.
6. Image the cells.
Spectral information and storage
Excitation/Emission (nm)358/461
Standard filter setDAPI
EVOS® Light CubeDAPI
Storage conditions≤–20°C



Protocol tips

  • DAPI is a known mutagen and should be handled with care.

Drosophila melanogaster embryo staining
Drosophila melanogaster embryo staining. Wild type (Canton-S) Drosophila melanogaster embryo exhibiting microtubule staining (green fluorescence), denticle band staining (red fluorescence), and nuclear staining (blue-fluorescent DAPI).