GFP and immunofluorescence

Gunther Rezniczek gr at
Mon Apr 7 10:23:56 EST 1997

Stephan Geley <s.geley at ICRF.ICNET.UK> wrote:
> I am using GFP as cotransfection marker in transient transfection assays
> which gives me a bright signal in live cells. However, when I try to fix
> cells for immunostaining I always loose the GFP signal. So far I've used
> Methnanol or Methanol/Acetone or Paraformaldehyd followed by .1% TX100
> without any success. Can anyone out there in the cybercommunity give me
> some advice? 
> Thanks anyway,
> Stephan

I have also made the observation that the GFP fluorescense is lost in cells
fixed with methanol.  In this case I expressed the GFP directly from an
unmodified pEGFP-N3 or pEGFP-C1 vector (Clontech).  However, when
expressing the GFP as a fusion protein (with the GFP at the C-terminal end
of the construct), I was able to fix the cells with methanol without any
loss of signal.  I have used about 10 different fusion proteins so far and
it worked with all of them.  You might try to just clone any protein in
front of the GFP.

Hope this helps,


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