Depending on the tissue, GFP fluorescence is fine after fixation with
(para)formaldehyde in buffer.  Try it and see if it's OK in your tissues.
You may be able to fix fairly lightly - 1-2% formaldehyde for a few minutes,
just enough to permeabilise the membranes without wrecking internal
structures.

If you're looking at Arabidopsis roots, you should be able to see the nuclei
in the transmitted light channel without staining, unless you're looking at
cells in the stele.  Leaves are a bit more tricky, but you can see them in
Arabidopsis epidermal cells.

Many of the syto dyes stain plant nuclei but they tend to stain quite a bit
of other stuff as well, and also mitochondrial and chloroplast DNA.  They
also seem fairly toxic, but I haven't used them extensively and others may
have had more success with living tissues.  Draq5 works to some extent
though it doesn't seem to penetrate intact plant membranes or walls very
well, but I haven't explored this much.

If you leave tissues in propidium iodide long enough, it will eventually get
into the cells, though their membranes may be a bit compromised.  You can
then image PI fluorescence separately from chlorophyll, and from GFP.

good luck,
cheers,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
E [log in to unmask]


On 18/05/10 7:15 AM, "Joel Sheffield" <[log in to unmask]> wrote:

> Have you tried Syto 9, from Invitrogen?
> 
> -------------- Original message ---------------
> We have researchers seeking to stain the nuclei in live plant
> tissues. DAPI will not penetrate, even in root material.
> Intentionally tearing or delaminating leaves will allow DAPI
> penetration for a short distance, but is too invasive to the living
> cells. 
> 
> Has anyone tried ethidium bromide on whole, plant tissues? How about
> some of the other nuclear stains?
> 
> The ultimate goal is double and triple staining with GFP to image on
> a laser confocal microscope. Fixation would be okay for one of the
> projects, but evidently GFP fades drastically when fixed.
> 
> Anyone have experience or suggestions?
> 
> Much appreciated,
> ~Gregg (on behalf of others)
> 
> Gregg Sobocinski
> Microscope Imaging Specialist
> University of Michigan, MCDB Dept.
> Ann Arbor, Michigan
> USA
> 
> 
> --
> Joel B. Sheffield, Ph.D.
> Biology Department, Temple University
> 1900 North 12th Street
> Philadelphia, PA 19122
> [log in to unmask]  
> (215) 204 8839, fax (215) 204 0486
> http://astro.temple.edu/~jbs