Green fluorescent protein/fixation

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From:Gayle Callis <> (by way of Marvin Hanna)
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GFP should be fixed with formalin or paraformaldehyde, acetone is probably
ruining the protein.  GFP is sometimes sensitive to solvents, and
especially to an initial fixation.  There are some who have fixed with NBF,
processed into paraffin with success something we are going to try with the
green glowing mice.
GFP is also sensitive to other things, enzymes, pH, temperature, etc. and
we totally lost it with a hematoxylin counterstain.  I have not seen it
used with any peroxidase or alk phos methods, but someone may be  having
success with those immunostaining methods combined with GFP.

Any fixation with acetone caused total loss of the GFP fluorescence in our
(on frozens) so we fix with 2% PFA in Dulbeccco PBS for 5 min, then run
with a another antibody fluorescent marker (usually Strepavidin Alexa 546,
a TRITC replacement) from Molecular Probes used with a biotinylated
secondary or biotinylated primary.  We now avoid any solvents for fixation.

The hydrogen peroxide might also be a factor, that can be tested on two
slides where cells with GFP are fixed with PFA, then one treated with the
peroxide, the other not treated, look at them at that point, no other
staining.  Actually, this could be a test at every step of a protocol, whew
- lots of work!   There is a great deal of literature on GFP, much of it in
physical chemistry journals. Clontech has a Living Colors Manual for GFP
users, filled with references.  It would be a good idea to get the freebie
from them since you are working with their GFP, it is worth its weight in

If the GFP fluorescence is lost, anti-GFP antibody could help out, but in
my estimation, if you are going to do that, you have somewhat defeated the
purpose of using GFP directly and its wonderful fluorescent properties.

Gayle Callis
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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