We have viral vectors that co-express GFP with other proteins in rat
tissues & routinely embed them in hydroxyethyl methacrylate
(Technovit 8100 from Heraeus). We use the protocol detailed in the
product literature and find that we are able to observe GFP
expression quite well after this procedure. I expect that we lose
detectable signal in areas where the expression was low, however in
some areas, we see very high levels of fluorescence. Briefly, the
protocol calls for overnight fixation in 4% paraformaldehyde,
overnight wash with sucrose-containing PBS, and a short acetone
dehydration prior to infiltration and embedding.

In these tissues, we are also able to see strong GFP expression
patterns if we fix in 4% paraformaldehyde prior to embedding in OCT
for cryo-sectioning. We have assumed (perhaps naively) that the
freezing without fixation causes the cytoplasmic GFP to leak out of
the cells when they're rehydrated and processed for
immunofluorescence. Whatever the mechanism, we do lose fluorescence
if we freeze tissues or cells directly. Fixation seems to help
preserve the fluorescence.

I have also stained fixed tissue with propidium iodide after triton
treatment and haven't noticed loss of GFP signal in these tissues.

Good luck!
Shawna


>Dear Rosemary,
>
>Would you please keep us up to date on your attempts to dehydrate in GMA etc.
>I have been trying gentle versions of most of the standard dehydration and
>embedment protocols with very little success.  I have noted that both fixing
>and freezing in an attempt to cryosection GFP and YFP expressing tissues seems
>to quench fluorescence entirely as seen by epifluorescence but that when
>viewed by confocal there is enough left to form a reasonable image.  Have you
>tried looking using the confocal even though the FP seems to have gone away?
>One other suggestion from the lab here was that you find another profession.
>I think that's just from the frustration of trying to clear tissues and still
>see the GFP expression pattern.  We are really very happy to hear that you are
>doing all of this work and we wish you well.
>
>Sincerely, John.
>
>Rosemary White wrote:
>
> > 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]
>
>--
>C. John Runions, Ph. D.
>Department of Plant Sciences
>University of Cambridge
>Downing St.
>Cambridge
>UK   CB2 3EA
>
>email: [log in to unmask]
>phone: (01223) 766 545
>
>http://www.plantsci.cam.ac.uk/Haseloff/JohnRunions/Home.html