Very strange. In our hands, paraformaldehyde kills cells very quickly
which is generally all that is needed for PI to enter the cells (it is
often used as a dead cell indicator). Overnight in PI seems pretty long.
What concentration are you using?
 
The ethanol stuff I've never tried but I guess I would worry about DNA
precipitation with ethanol. What concentration are you using. Also, PI
does stain nucleoli intensly. It also stains RNA. In fact, any double
stranded nucleic acid is likely to get stained, which could explain the
cytoplasm.
 
________________________________________________________________________________
 
 
Paul Goodwin
Image Analysis Lab
FHCRC, Seattle, WA
 
On Thu, 6 Jun 1996, Tom Phillips wrote:
 
> I am trying to stain a monolayer of cells growing on a 12 mm glass
> coverslip with propidium iodide.  We fixed the monolayer with either
> ethanol or 2% paraformaldehyde and then stained overnite with 5 ug/ml
> propidium iodide, rinsed,  mounted using mowiol, and examined using an
> MRC600.  The ethanol fixed cells had intensely stained nucleoli but the
> nuclei themselves were still dark against a lightly stained cytoplasm.
> QUESTION:  Can anybody tell me why the nuclei themselves aren't staining?
> Nuclei in 1 um JB-4 methacrylate sections of paraformaldehyde fixed cells
> stain beautifully and homogeneously using 5 ug/ml PI.
>
> The paraformaldehyde fixed cells had a general light diffuse staining with
> no specific staining of the nuclei or nucleoli.  I assume this is because
> the paraformaldehyde didn't permeabilize these cells.  Alternative
> explanations welcomed.
>
> Thanks in advance for any advice or comments.
>
>
> Thomas E. Phillips, Ph.D.
> Associate Professor of Biological Sciences
> Director, Molecular Cytology Core Facility
> 3 Tucker Hall
> University of Missouri
> Columbia, MO 65211
> (314)-882-4712 (voice)
> (314)-882-0123 (fax)
>