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

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