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

From:"Jamie E Erickson"

                 I have had experience with looking at vectors in Mouse 
tissue years ago using EGFP as a report gene and had no trouble 
fixing/processing/embedding these samples in paraffin on my standard 
protocol. EGFP is very stable to these harsh conditions. I presented this 
data at the NSH poster session back in 99. Here was the conclusions, If 
you would like a copy of the methods or the poster contact me and I'll 
send you the Powerpoint poster. 
The fluorescence of EGFP was still visible after routine fixation, 
processing, and 
paraffin embedding using ethanol and methanol as the dehydrating agent. 
For two of the three fixatives, EGFP fluorescence was still visible after 
being in fixative 
for as long as one month.  A decrease in fluorescence was observed using 
the zinc-based fixative (Z-fix),
 compared to formalin or paraformaldehyde fixation. The fluorescence 
intensity decreased with increasing length of fixation.  EGFP fluorescence 
appeared optimal using 10% NBF at the12 and 24 hour timepoints.  Methanol 
processing appeared to better preserve the EGFP fluorescence using the 
zinc-based fixative (Z-fix),and at the later timepoints.  These data 
suggest that routine histologic procedures and the use of common fixatives 
such as 
10% neutral-buffered formalin can be used in the analysis of 
adenovirus-EGFP expression in vivo.  Thus, the use of EGFP as a reporter molecule is an attractive 
alternative to -galactosidase for expression analysis. 
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