Dear Gayle,

  Congratulations to you (and the other responders) about GFP fixations
successes. I have been following GFP for several years and have accumulated the
following tips [I'm pasting these from a larger microscopy document so some of
it may read a little weird]
Mike Moser wrote that “Alcohol fixation will rapidly destroy GFP fluorescence. 
I use 1/20th volume of formalin for 5 minutes at RT.  Cells will remain
fluorescent for several days stored in PBS at 4 C.  WT GFP is essentially
useless.  Human codon bias optimized S65T (or other enhanced) GFP is highly
recommended for FACS w/ human cells.” (Mike Moser, Department of Pathology, U.
Washington, Seattle, WA. Fluorescent Protein newsgroup, 4/24/97).

Chalfie et al (1994) mention that using nail polish to seal coverglasses to
slides causes rapid loss of GFP fluorescence.

See also Chalfie et al (1994), Clontech, Quantum Biotechnologies, Life
Technologies and Invitrogen literature for other hints. The upshot is that GFP
works best on live cells!

Brock et al (1999) studied GFP-EGFR in living an fixed mammalian cells. Their
concern was whether fixatives and/or mounting medium might cause apparent
redistribution of membrane proteins (such as EGFR, a growth factor receptor).
They report that,   
"Paraformaldehyde (PFA) fixation with subsequent mounting in the antifading
agent Mowiol, but not in Tris- or HEPES buffered saline, led to a partial
redistribution of the EGFR from the plasma membrane to the perinuclear region.
The redistribution was confirmed with the GFP and EGFR immunofluorescence. The
in vivo distribution in Mowiol mounted cells was preserved if cells were
treated with a combined PFA/methanol fixation procedure, which also retained
the fluorescence of soluble GFP… The combined PFA/methanol protocol is
universally applicable for the fixation of transmembrane and soluble
cytoplasmic proteins and preserves the fluorescence of GFP."

  Search Medline   http://www.ncbi.nlm.nih.gov/PubMed/    for the exact
references and to read the abstracts. The Brock paper is from one of the best
cell/microscopy labs in the world (Tom Jovin's lab), so there is a high trust
factor in the Brock paper.


  By the way, I have also been following the double immunostaining thread. I am
interested in hearing (by direct email to george.mcnamara@spectral-imaging.com)
from anyone interested in reliably visualizing double and triple "staining",
where the staining can be fluorescent (there are now four colors of GFP's
available) or chromogens or standard bright-field stains (i.e. H&E&DAB&BCIP). I
have imaged triple immunostains slides from both Vector Labs, and from a
customer, as well as a 5-immunostained slide from DAKO (breast carcinoma
antigen panel ... yes the section survived 5 rounds of staining ... my thanks
to April and Dave of DAKO for going to to the trouble of making this slide).
You'll have to talk with Vector, DAKO, etc., about how to make the slides.


Sincerely,


George



>Date: 28 Apr 1999 12:46:31 -0500 
>From: Gayle Callis <uvsgc@msu.oscs.montana.edu> 
>Subject: GFP/fixation/paraffin sections
>I had (much to my happiness!) two replies from people who had used 
>GFP with SUCCESS in formalin fixed tissues/paraffin processing. 
>Could it be that the GFP they are using is a stable form (heat stabile, 
>resistant to solvents?). If so I am hoping they share the specifics 
>of what was done, exact GFP used (there are chimeras out there!) and 
>some of the processing times/solvents/paraffin. 
>At least we had one thing in common, formalin fixation of tissues, time 
>of fixation may be a big factor also.
>ladies, are you willing to share these goodies, it would be a big help.
>Gayle Callis



George McNamara, Ph.D.
Applied Spectral Imaging, Inc.
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