[Histonet] GFP again

Caroline Bass cbass <@t> bidmc.harvard.edu
Mon Apr 25 12:21:41 CDT 2005

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.


Caroline Bass

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