Hello, Young Jik,

I have worked for several years on endogenous CFP, YFP, RFP and GFP in
transgenic mice as well as neurons (both primary cultured and in vivo) that
are transfected via plasmid vectors to express YFP, GFP and dsRed and there
are some important issues that I discovered that I want to share.

1) Here is how I prepare my brain sections: mice/rats are perfused with cold
PBS/4% para is made fresh as everyone has pointed out.  I use the brown
ampules from EMS as well - but we never save them (we always use them
fresh).  I've found out that the brightness of the GFP is significantly
reduced if the perfusion is not as good.  

2)  The brains are postfixed in 2% para overnight.  Over fixation increases
background fluorescence especially in the YFP range, but I personally have
not observed overfixation reducing the signal (perhaps this is because I use
fresh PFA).

3) ** I've found that using a cryostat, rather than a vibratome
significantly reduces the intensity when I view the sections without
immunostaining.  I've done numerous testings in the lab to confirm this - I
think the brains being frozen somehow affects the GFP structure, because the
brains are otherwise prepared identically when I test them side by side.

4)  XFP's have to be hydrated to be visible as has been elucidated by one
posters.  This is why a sectioned brain that is dried will not show any GFP+
cells, but once it is hydrated even with PBS, will fluoresce.  Therefore, a
wet mounting medium is important to use.  I currently use Prolong Gold from
Invritrogen - when the agent cures, it solidifies a little, but still has to
be sealed with nail polish because it does not solidify completely (I am
assuming this last part).  I used to use Vectashield which was also good,
but it remained completely wet - I have had not such experience with
Fluormount G - but I think mine had been in the lab for some time so maybe a
fresh one may work well.

5)  Lastly, as another poster mentioned, you could use a GFP antibody to
bring out the signal - however, I think it is difficult to quantify
gene-expression based on the intensity of signal (with or without
immunostaining) because the intensity can be lowered due to many factors
mentioned above (that is, if you are working with a whole animal, and
obtaining the sections - perhaps this is less of a concern in a cultured
prep).  As cumbersome as it may be, perhaps doing a pilot RT-PCR with GFP
fluorescent cells may be enough to establish if the intensity is a reliable
predictor of your gene-expression using your protocol, because however you
are preparing your cells/sections may or may not be a good method.

I hope this helps and good luck!

Won Yung Choi

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Won Yung Choi, Ph.D.  
Research Scientist      	
Department of Molecular Therapeutics
New York State Psychiatric Institute/    
Department of Psychiatry                          
Columbia University                                      
1051 Riverside Drive, NYSPI Unit 62        
New York, NY 10032 (212) 543-6058 
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