[Histonet] fluorescent proteins (RFP and GFP) native fluorescence

From:Caroline Bass

Hey guys,

I was hoping someone here could give me some advice on their  
experience with various fluorescent proteins.  I need a good marker  
for my viral vector.  I have a humanized GFP, RFP and EGFP.  I would  
like to inject the vector into the tail vein to see which tissue  
lights up.  My hGFP variant is not very strong in vitro, but both RFP  
and EGFP work well.  I have heard that GFP quickly disperses when  
mounted on slides.  However, I have seen some papers that use native  
EGFP fluorescence with cryosections and they don't have problems.   
Ideally I would use floating tissue sections of 40 microns as I have  
easy access to a sliding microtome.  I have heard that RFP does not  
work with fixation at all.

I am open to any sort of advice.  Could someone recommend a protocol  
for visualizing native fluorescence with either EGFP or RFP?   
Specifically, whether to fix or not, the thickness of the section,  
floating sections vs. slide mounts, etc.

Also, if there is another fluorescent protein available that you  
could suggest I would like to hear about it.  I have heard of yellow  
and blue fluorescent proteins as well as a monomeric form or RFP.  I  
am also open to using a "universal" fusion protein, for example actin- 
GFP if it solves the problem of GFP native fluorescence.



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