[Histonet] GFP, the ongoing dilemma with DsRed included

[Gayle Callis]

Dear Caroline,

You wrote:
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.

***We recently undertook a similar project with undecalcified murine nasal 
turbinate and small intestine tissue sections from mouse.

In order to do immunofluorescence staining for a CD marker or dendritic 
cell, we were required to fix with acetone or acetone/alcohol, which 
immediately killed the eGFP and it failed to glow. This was also going be a 
problem with DsRED, a bioluminescent protein from Coral rather than jellyfish.

One major problem is increasing autofluorescence in tissues by using 
formalin or paraformaldehyde fixatives. We could never get the best of both 
worlds with eGFP or DsRed.

Final solution to the problem:

With DsRed, unfixed fresh snap frozen tissues were cryosectioned at 5 um, 
mounted on Erie Plus Gold slides (a plus charged surface for extra holding 
power),and air dried for 30 min or more in front of a fan at RT.  Air dried 
sections were rinsed one slide at time to prevent unfixed section loss with 
2 changes PBS gentle agitation.  We wanted to get rid of OCT. A coverslip 
was mounted with Prolong Gold antifade mounting media, ready to use 
containing DAPI for DNA/RNA.

Results:  spectacular DsRed where it was supposed to be, with nuclei 
counterfluorescing a lovely blue from DAPI staining DNA/RNA.  The mounting 
media is a hard set.  The only autofluorescence is natural, but does NOT 
interfere with the brighter signal of DsRed, and would not interfere with 
eGFP either.

We are going to do this with eGFP next week to have green fluorescence with 
the DAPI stained nuclei.

I have a review of autofluorescence I am going to attach to you.  For more 
on eGFP and DsRed, go to Clontech website and download their Living Colours 
Manuals for these proteins.

When you get to immunostaining, say for for a CD marker along with 
eGFP,  we did our acetone/alcohol fixation, but detected eGFP with Goat 
antiGFP from Rockland (a superb antibody that is NOT expensive!) and came 
back with Donkey antiGoat-FITC, along with a biotinylated CD marker 
antibody and Strepavidin Alexa 555.  This worked well for double 
fluorescence with the CD marker.  As for DsRed, the antiDsRed antibody is 
pricey, so doing IFA staining for GFP has been more cost effective.