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nuclei staining and immunolocalization - (Nov/29/2007 )

hi everyone,
I'm pretty new with the use of fluorescent tag to visualize proteins and so on..
I made an immunoloc experiment using antibodies coupled to FITC, and wanted to visualize the nuclei using DAPI staining, but the UV laser is down....
Someone suggested to stain nuclei with a visible dye such as CYTOX visible wavelength DNA dyes (INVITROGEN-- formerly
Molecular Probes). Do you allready used that dye, or do you have a simple way to do the same thing with a cheaper dye?
thanx a lot


You could certainly use cytox dyes to visualize the nucleus and they are very easy to use. The only problem you might have is that this takes up one of your channels for imaging. For example, if you use cytox green for DNA staining, you can't use Fitc. They are both green and can only be imaged on one channel. I don't know how many channels you have available on your system but ours is only 3. If the UV goes down that means all we have is green and red. If I were you I'd start screaming and hollering to the boss to fix the UV.


Assuming your cells a fixed and permeabilized, you could use propidium iodide on the red channel - it's relatively cheap and super bright. Wash cells in a 15 ug/ml PI solution in pH 6.8 citrate for 5 minutes and then wash for 5-10 min in running DI. Depending on the level background you may also have to treat the cells with RNase, but that is usually not a problem for my cells.


propidium iodide seems great, but I'am doing this on animals than need to be counterstain with evans blue due to their autofluorescence, Ithink it will totally mask the pi staining.