It sounds like you might be a) using too much fluorescent dye; or b) not washing the cells thoroughly enough after staining. It's quite common to get non-specific staining when dye concentrations are relatively high.

I would agree with a possible dilution problem. In the lab we dilute DAPI at 1/40,000 too 1/50,000 in PBS and let it for a few minutes on the cells. Followed by several washes.

I would suggest, if the money not an issue to buy mounting media that has Dapi (we use the one from Invitrogen), just because it does work all the time in both sections (human and mice) and cells.

But again do you have any staining at all in the nucleus or is it stuck in the membrane?? are you using only Dapi or in conjunction  with another antibody (in this case your protein of interest), the question I have is what is the channel you are using, could it be that there is overlap between the 2 channels if they are close enough. The other issue are your cells permeabilized ???

Thanks Dear Aicha,

in this case, the only dye is DAPI and nothing else. the thing is when I wash the cells DAPI is not in membrane any more but it looks it shows just the damaged cells nuclei!! I mean even though there are many healthy nuclei around, DAPI doesn't show them at all. Do you think if I increase the incubation time to half an hour or so it can help?

Best,

I think what you need to try is to permeabilize the cells, it looks to me that the Dapi at least in the formulation you are using is not getting to the cells (10 to 20 min with PBS that contains 0.05% Tween and 0.3 Triton). 

There is also another counter staining for the nucleus that you could also try: Propidium Iodide (again this one is all in one;  mounting media plus the stain PI).