DAPI Protocol for Fluorescence Imaging

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

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.

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).