DAPI (4′,6-diamidino-2-phenylindole) is a fluorescent stain that binds strongly to DNA. It is commonly used in fluorescence microscopy for visualizing cell nuclei because its fluorescence is enhanced when bound to double-strand DNA.
The protocol:
For staining of nuclear DNA in common adherent cells, a DAPI concentration in the range of 1-10 µg/mL is usually sufficient. However, the optimal concentration can depend on several factors: cell type, specific application and the other components of your staining protocol. An often-used protocol is to dilute a DAPI stock solution (often 5 mg/mL in water) in your buffer of choice (often PBS) to a final concentration of around 1-5 µg/mL.
The cells are then incubated with the DAPI solution for a few minutes (commonly 5-10 minutes). As with any protocol, it’s important to optimize the conditions for your specific application.
Why we do not recommend DAPI in mounting media
While some researchers do use a mounting medium that already contains DAPI, it’s not always the best choice for every experiment, and here’s why:
1. Control over concentration: By staining DAPI in a separate step, you have more control over the concentration of the stain. This allows you to optimize the staining intensity for your specific experimental setup, something that isn’t possible with premixed mounting mediums.
2. More consistent staining: Staining in a separate step can also lead to more consistent staining across the sample. In contrast, the DAPI in mounting medium can sometimes produce uneven staining, particularly in thicker specimens or samples with uneven surfaces.
3. Timing: If you use a mounting medium with DAPI, you’re limited to staining at the very end of the protocol. By staining separately, you can choose to stain at a different point in your protocol if it suits your experiment better.
4. Mounting media variations: Not all mounting media are compatible with all types of specimens and fluorescence microscopy techniques. If your experiment requires a specific type of mounting medium, you might not be able to find one that also contains DAPI.
That being said, using a DAPI-containing mounting medium can save time and is often sufficient for many experiments. It largely depends on the specifics of your experimental setup and the level of control and optimization you require.
