[Histonet] rfp in cryosections

[Richard Pattison]

Hello,
I am currently making cryosections of transgenic tomato fruit expressing 
mRFP 
<http://www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/Product-Spectra.mRFP.html> 
but am having some problems visualising fluorescence with a confocal. I 
realise fluorescent proteins work best in live cells but I have read a 
few reports of direct GFP detection in fixed cryosections.  When I view 
hand sections of the fruit I can easily see the flourescence with no 
autofluorescence in my wild type control. However, with my cryosections 
the autofluorescence increases and the RFP fluorescence is so diminished 
i can't distinguish it. I've tried three different fixatives (in order 
of most to least autofluorescence: 4% formaldehyde/0.25% glutaraldehyde 
in PBS, 3:1 ethanol:acetic acid and 1:3 ethanol:acetic acid), followed 
by infiltration with 20% sucrose in PBS. I embed in OCT and use the 
cryojane tape transfer system to transfer the sections to slides. After 
sectioning at 14 microns I dry the slides at room temperature before 
viewing. I assume some part of the fixation process (and/or the cryojane 
tape transfer process) is leading to autofluorescence in the RFP range. 
Does anyone have any suggestions for minimizing this?
Thanks in advance,
Richard

-- 
Richard Pattison
Boyce Thompson Institute for Plant Research
Tower Road
Ithaca, NY, 14853-1801
USA
Phone: +1 607 254 8757
rjp226 <@t> cornell.edu
http://bti.cornell.edu/CarmenCatala.php