Dear people,

We are trying to localise the nucleus in barley aleurone protoplasts
transiently expressing GFP (they are transfected with Agrobacterium one
afternoon and observed the following afternoon).  However, none of the
membrane permeant DNA stains we tested - DAPI, and all of the red and
orange SYTO dyes - get into these things.  These protoplasts are "terminal"
and don't make much of a wall, and will burst even 24-36 h after isolation,
so we think the plasma membrane is the problem.

So we tried fixing the cells, and/or permeabilising the membrane, but in
every case, when the nuclear dyes got in, the GFP died.  Fixed cells with
low or high paraformaldehyde, with and without low or high Tween20 or DMSO
or TritonX100, or just added varying amounts of detergent.  Also tried
adding EGTA to "loosen up" any polysaccharides secreted by the cells.
Tried killing the cells in other ways - with azide, for example.  As soon
as the cells were dead, the dyes got in, and non-permeant dyes also, like
propidium iodide, but in every case the GFP had gone.

Any ideas, anyone??  These protoplasts are packed full of starch grains,
small vacuoles, other vesicles and dense cytoplasm, so the nucleus is not
readily seen with DIC, though there is a large region that doesn't have
organelles and that is most probably the nucleus.  However, we need to know
for sure!

As a last resort, I plan to stick the protoplasts down onto a slide, image
the GFP, wash through with fixative and DNA stain, then image again.
However, if anyone has a neat trick that would allow us to image GFP and
DNA simultaneously, we'd be most grateful.

TIA,
cheers,
Rosemary


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

phone  61-2-6246 5475
fax       61-2-6246 5000
email   [log in to unmask]