Hi John,

Our viral vectors express GFP at a very high level by 36 hours
post-infection. At the earlier timepoints, we have difficulty
identifying infected areas by epifluorescence, which we have always
attributed to attenuation of fluorescence in the fixation protocol. I
haven't tried looking at the early timepoints by confocal, which is a
great idea! We feel that the signal we are seeing in infected tissues
is specific, since we can see positive cells alongside negative
cells, it localizes appropriately in the cells, and it's only
observed in the FITC filter set (unlike much of the autofluorescent
signal in these tissues which is visualized with both the FITC and
the rhodamine filters)

I meant to provide a reference for embedding the GFP-expressing
tissues in Technovit 8100. We got the idea from this reference:

Ohta H, et al. Regulation of proliferation and differentiation in
spermatogonial stem cells: the role of c-kit and its ligand SCF.
Development. 2000 May;127(10):2125-31.

Your idea about the protein being quenched by freezing is
interesting. I haven't tried to look for the protein in frozen
tissues using an anti-GFP antibody, but would be interested to know
if anyone on the list has. It would be nice to know whether it is
still there but not functional, or if it is leaking out during the
freezing process. Incidentally, we also have observed GFP loss when
we do "touch preps," which is a messy technique in which the tissue
is cut into pieces and the cut ends are touched to a poly-l-lys
slide, dried down, then rehydrated and processed for
immunofluorescence. We have assumed that the disruption of the cell
membrane which occurs with drying was leading to loss of the GFP from
the cell, but again, we haven't tested these ideas. We have just
avoided doing anything with the unfixed tissue.



>Dear Shawna,
>
>I am astounded that you can acetone dehydrate and embed in HMA and
>still see GFP
>fluorescence.  This is good news and I will try again with an
>acetone series.  As
>far as freezing goes, I was unsure about where the GFP was going so I tested
>freezing and immersion in OCT seperately.  Freezing seems to
>irreversibly quench
>GFP fluorescence whereas immersion in OCT is not so bad in the short
>term simply
>because it doesn't penetrate very quickly into the tissues.  Root
>hairs lost their
>fluorescence first in OCT and other peripheral tissues showed signs
>of dehydration
>and quenching of fluorescence after a couple of hours.  When I check
>cryosectioned
>material, there is a low level of GFP and YFP fluorescence suitable
>for screening
>by confocal microscopy but it is not visible by epifluorescence
>steromicroscope.
>
>Shawna Fleming wrote:
>
> > 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
>
>--
>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