Perhaps your PI has gone "bad".  If it has been in aqueous solution for
too long, or left in the light strange things could happen.  I remember
being mystified once and finding that a new batch of PI fixed
everything.  The ETOH or paraform should be fine for perm.
 
Tom Phillips wrote:
>
>  I am using 5 ug/ml PI for all steps.  The fixation was for 2 days in
> either 100% ethanol or 2% paraformaldehyde for 2 days (long fixation time
> for convenience). I have previously tried shorter fixation times with a
> ethidium homodimer and saw a similar phenomenon where only some of the
> cells were permeabilized by the fixation step but 100% were permeable if
> fixation with PF was followed by an organic solvent or detergent.  I am not
> really bothered by the background staining of the cytoplasm which I assume
> is due to RNA.  But the nucleus itself is dimmer than the cytoplasm and
> essentially unstained.
>
> >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)
> >>
>
> 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)
 
--
 
    _______________________________________________________
   / Michael J. Herron,  U of MN, Dept. of Dermatology    /
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 / 612-625-8935  Box 98  UMHC, Mpls MN 55455            /
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