[Histonet] GFP again

Mon Apr 25 12:55:57 CDT 2005

Caroline,

I am pretty much a novice at the GFP but have done some work on this. I can
tell you that the protein is very soluble and does not survive standard
fixation with NBF when working with frozen sections. There is a nice paper J
Histochem Cytochem. 2003 Mar;51(3):401-4 that discusses this and proposes a
vapor fixation. 

We have had good luck with formalin fixed paraffin embedded tissue using
anti GFP antibodies. In my hands there is considerable variation in the
sensitivity and signal /noise ratio. My suspicion is that due to the
solubility of the protein perfused specimens may be preferable. But I have
not tested this. 

We have started a project with grafting of GFP expressing tissue into a non
GFP expressing mouse and I can tell you that seeing the GFP in the fresh
tissue is not as obvious as some of the literature would lead one to
believe.

I am still working on this so I look forward to replies from others more
knowledgeable and experienced than I.

c

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317

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-----Original Message-----
From: Caroline Bass [mailto:cbass <@t> bidmc.harvard.edu] 
Sent: Monday, April 25, 2005 10:22 AM
To: <histonet <@t> lists.utsouthwestern.edu> <histonet <@t> lists.utsouthwestern.edu>
Subject: [Histonet] GFP again

Hey Guys,

I have been searching the internet and all sorts of forums, including 
histonet trying to find the answers to my GFP questions.  Essentially, 
I have a viral vector that I would like to inject into the liver which 
expresses EGFP.  I would like to have the easiest way to determine if 
it works or not.  I have heard that it is virtually impossible to 
detect native fluorescence with GFP, and that frozen cryostat sections 
completely lose the signal.  Am I missing something here?  There is a 
paper that is essentially doing the same thing that I would like to do, 
and they have enough signal that they can see it in the intact animal 
with a UV lamp, or in 6 micron cryosections without fixation or 
perfusion.  According to everything I read, this shouldn't work.  Does 
EGFP produce a stronger signal that can be seen in this way?

The paper is:  Nature Biotechnol 2005 Mar; 23(3):321-8.

Any suggestions on how to process the liver such that I can see the 
native EGFP signal would be greatly appreciated.  I have access to a 
cryostat and a microtome.  I can freeze the tissue, perfuse the mouse, 
whatever it takes.  I would prefer not to use immunostaining initially, 
as I have to section through entire lobes to visualize where the virus 
diffuses, etc.  I can use an antibody once I get a rough estimate of 
where the signal is, but we don't have enough money to immunostain 
every section.

Thanks,

Caroline Bass

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