Dear Alice,

alice schmid wrote:
>        Not really sure what to do about the nuclear
>stains---but in drosophila we pretty much accept the
>fact that any fixation kills GFP fluorescence.

We've found that in most of the plant tissues we've tested (which isn't
actually all that many as yet), fixation in 4% paraformaldehyde in
phosphate buffer doesn't kill the GFP.  We wondered if these aleurone cells
had something particularly nasty stored in their vacuoles that chews up the
GFP and other proteins as soon as the vacuole membrane is breached.

If we try to embed our GFP-tagged tissue, it's dehydration in solvents that
does it - at about 95% solvent, the fluorescence goes.  We're currently
trying an old GMA method where you dehydrate directly in GMA monomer, and
also trying a very quick dehydration/infiltration in LRWhite, and so far,
there is still GFP fluorescence at the beginning of resin infiltration in
both cases.  We'll see how much longer it lasts.

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
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