Can I use DAPI and GFP at the same time? - Need advice on fluorochromes for confocal microscopy (Feb/20/2008 )

Hi all,

I'm planning to use confocal microscopy to look at the subcellular localisation of a GFP-tagged protein. Ideally I'd like to use DAPI to stain the nucleus, but I'm worried about its wide emission spectrum.

It looks like DAPI's wide emission specrum will make its signal overlap that of GFP. If emissions from DAPI are picked up in GFP's channel, this could make it look like my GFP-tagged protein is localising to the nucleus when it actually isn't.

Does anyone have experience using the two together? Do I need to worry that the DAPI signal will be picked up in the GFP channel?

If so, can anyone suggest an alternative? I'm considering RNAse treating my cells then staining with PI, but any advice would be really helpful!

Thanks,
Melville.

Hi,

I think you should be able to use them together. If DAPI staining is only to mark the nucleus, you can use DAPI as diluted as possible.

Maybe you give a try on your sample first and then you can decide what next to do. It also depends how strong the GFP signal is.

One should not have a problem with using DAPI and GFP together.

Thanks! As there are no known problems, I'll give it a try.

i'm assuming you're using dapi in vectasheild so you cant dilute it.
they are a bit close but if you use sequential scanning you should be ok (other wise the emitted dapi could exite the gfp and give false colocalisation)

dom

I took some cells which don't express GFP and stained them with DAPI. The nuclei light up as expected when looking through the DAPI filter, but look dark through the GFP filter. So I think I'm OK.

Thanks for the help all!

that makes no difference whatsoever unless your microscope is also using the same filters and separatly the way you looked at the tissue - dual exitation lasers are a whole different ball game

ie what kind of microscope are you using

dom

I've used it together and got a beautiful pic - dapi/gfp/alexa 555 secondary antibody against V5 tag. See attachment.