After having many problems with propidium iodide staining (cytoplasmic
staining) we were advised to use cold methanol fixation which worked well.
However we could NOT use formaldehyde fix at all.  Now we know that RNAse
incubation prior to staining does the trick and allows for a decent PI
staining in formaldehyde-fixed cells.
Michal Opas
U Toronto
 
On Wed, 22 Mar 1995, Richard Levenson wrote:
 
> Hi. I have been pottering about trying to optimize a staining
> protocol for formaldehyde, glutaraldehyde or unfixed tissue using
> eosin and propidium iodide.  I'm using aqueous formulations of
> eosin Y OR and 5 mics/ml PI and the results are adequate but not
> stunning under confocal.
> Has anyone a more detailed protocol (buffers, pH's, aqueous vs ethanolic,
> time, order of staining, species of eosin, etc.)
>
> The idea is to be able to get a really good look at a thick slice of
> tissue with optical sectioning in a little from the cut surface.
>
> Thanks,
>
>
> Richard Levenson, M.D.
> Depts. of Pathology and Computer Science,
> Duke University, and GRECC, Durham VA Medical Center
> Phone: 919-286-0411 x 6776
> Fax:   919-286-6823
> Duke beeper:  919-970-0287
>
> Mailing address:
> GRECC #182,
> VA Medical Center
> 508 Fulton St.,
> Durham, NC 27705
>