Hi again Shawna, I should have mentioned that when I was observing GFP quenching after freezing I was looking at fusions between the fluorescent proteins and nuclear or plant cell-wall proteins.
This is because we had previously observed non-localised GFP leaching from cells during procedures like this. My guess is that the GFP stays in place. I, therefore, am curious as well about whether
it retains it's antigenicity. Thanks for the reference.
John
Shawna Fleming wrote:
> 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.
>
--
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
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