problem with propidium iodide staining - (Aug/15/2007 )

Hi every one,

I am having problem with propidium iodide nuclear staining. I fix and permeablize the cells (adherent) and after primary and secondary incubation and washing steps, I add 1ug/ml PI and incubate for 30 minutes at RT.

The problems are:
1) not all the nucleus light up- there are only a few red dots in the nucleus (condensed chromatin?)
2) I can see the red color in the cytoplasm as well, which means the PI has stained the cytoplasm as well. (Please see attached file for the picture)

I have tried different concentrations of the PI but it didn't help.

Any suggestions?

Thanks a lot

-sayeh-

QUOTE (sayeh @ Aug 15 2007, 03:50 PM)

Hi every one,
I am having problem with propidium iodide nuclear staining. I fix and permeablize the cells (adherent) and after primary and secondary incubation and washing steps, I add 1ug/ml PI and incubate for 30 minutes at RT.

The problems are:
1) not all the nucleus light up- there are only a few red dots in the nucleus (condensed chromatin?)
2) I can see the red color in the cytoplasm as well, which means the PI has stained the cytoplasm as well. (Please see attached file for the picture)

I have tried different concentrations of the PI but it didn't help.
Any suggestions?
Thanks a lot

after fixation and perm, are you treating with RNAse A? P.I. will interculate into free RNA and make your cells light up like an xmas tree! If using P.I as a vital dye do not fix or perm, just add to media. Only dead cells will take up the dye, why live cells will expel the chemical.

hope this helps.

7's

-Lenti77-