DAPI Stock Preparation¶
- Version: v1.0
- Last modified: 2026-07-29
Preparation and storage of the DAPI stock. For the staining procedure itself, see Nuclear Staining (DAPI).
Product Information¶
| Property | Value |
|---|---|
| Product | DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) |
| Catalog | Thermo Scientific D1306 |
| Fluorescence | Ex/Em = 358/461 nm |
| Specificity | Binds AT-rich regions of dsDNA (nuclear stain) |
| Storage | Powder and stock at -20°C, protected from light |
1. Stock Solution (5 mg/ml)¶
- Bring the vial to room temperature and centrifuge briefly to collect powder.
- Dissolve in nuclease-free water to 5 mg/ml (e.g. a 10 mg vial in 2 ml). Dimethylformamide can be used if the powder is slow to dissolve.
- Aliquot 20--50 μl per tube and store at -20°C, protected from light. Avoid repeated freeze-thaw.
2. Working Solution -- 10 ng/ml¶
10 ng/ml, not 5 μg/ml
The long-standing 1:1000 dilution (5 μg/ml) is far more dye than nuclear staining needs, and at that concentration DAPI crosstalks into the 480 nm channel -- found in-house while setting up six-colour imaging, where it costs a channel needed for signal. 10 ng/ml stains nuclei perfectly adequately with no crosstalk.
Use 5 μg/ml only when DAPI is the only thing being imaged.
500,000x is not a single-step dilution. Go through an intermediate:
| Step | Dilution | From | Into | Result |
|---|---|---|---|---|
| 1 | 1:1000 | 1 μl stock (5 mg/ml) | 1 ml PBS | 5 μg/ml |
| 2 | 1:500 | 2 μl of step 1 | 1 ml PBS | 10 ng/ml (working) |
- Prepare the final dilution fresh and discard after use -- DAPI is not stable stored at this dilution.
- The 5 μg/ml intermediate can be kept short-term at 4°C, protected from light.
Where This Is Used¶
- Nuclear Staining (DAPI) -- staining procedure
- Cell Membrane Staining (WGA) -- combined DAPI/WGA mix
- Bill of Materials -- ordering information
Changelog¶
- v1.0 -- 2026-07-29 -- Split out of the DAPI staining protocol so the stock and its serial dilution live with the other stored reagents. Documents the move from 5 μg/ml to 10 ng/ml working concentration to eliminate 480 nm crosstalk in multiplexed imaging.