🚀 go-pugleaf

We have been employing GFP in confocal imaging here for a number of years in
a wide variety of specimens ( fly embryos,  yeast and bacterial cells and a
wide variety of mammalian cells).  It is a wonderful fluorescent tag for
tracing gene expression and molecular transport, and cell shape changes just
to mention a few of its uses.  But it does and can have problems in any
system from over expression, under expression, bleaching and leaching.  So I
thought I would pass on a few tips about its use for people who are just
beginning to employ it in their work.

GFP fluorescence is killed by dehydrating or fixation agents of ethanol and
acetone.  Its signal is also greatly reduced by formaldehyde fixation.  Long
term storage of samples in aldehydes will wipe it right out.  This will also
increase autofluorescence.   This type of storage is not wise in regards to
maintaining antigenicity of many proteins etc.  If you are looking for GFP
signal in specimens Employ freezing and cryosectioning instead of fixation
of tissues and embryos etc. If you are unable to do this then it requires
that you probe with an anti-GFP antibody.  The best direct imaging of GFP is
obtained in living tissue and cells.
For fixation employ low percentage paraformaldehyde for the shortest periods
of fixation as possible( 5- 10 minutes for cells).  Samples should be
mounted in glycerol based mounting medium.  I have found 90% buffered
glycerol to be best.  Avoid using nail polish as sealant of coverslips.  In
a short time this will wipe out your GFP signal.  Also if your GFP is not a
fusion protein that gets fixed in place, you will lose all signal upon any
permeabilization by detergents and freeze thawing.

For imaging in living cells and embryo's use the least exposure times you
can get away with as continued exposure can rapidly bleach your GFP.   The
addition of super radical scavengers in cell cultures systems is a good idea
to help slowing of bleach rate.  This can be an advantage if you are looking
at return of expression.

Good luck.

Joe Goodhouse
Confocal / EM Core Laboratory
Department of Molecular Biology
Princeton University
jgoodhouse@molbio.princeton.edu <mailto:Jgoodhouse@molbio.princeton.edu>
609-258-5432
Visit us at: http://www.molbio.princeton.edu/facility/confocal/index.html
<http://www.molbio.princeton.edu/facility/confocal/index.html>