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GFP detection

Carrington CARRINGTON at BIO.TAMU.EDU
Sat Nov 11 13:14:33 EST 1995


Examining GFP after particle bombardment of leaf tissue can lead to many 
hours of viewing pleasure and extensive bonding of lab personnel around the 
microscope.  All of our applications involve analysis of GFP fusion proteins 
that localize to the nucleus, to vesicles, etc.  We've found a number of 
tricks that enhance the GFP experience:

1. Bombard leaves in the afternoon or early evening and incubate tissue in 
continuous light overnight.  If detached, place leaves in a petri dish with 
water.  View your samples early the next morning.  

2.  To quickly assess the numbers of cells expressing GFP, you can scan a 
leaf by placing it dry on a slide and using an FITC filter set (at 10x 
magnification).  Although commonly cited, the FITC filter set at low mag with 
plant tissue has a few problems.  First, the intensity of GFP fluorescence at 
low magnification is just plain low, even with a strong promoter and 
expression cassette.  And second, the red autofluorescence from the leaf can 
partially obscure the GFP signal.  To circumvent these problems, we typically 
scan the tissue using a broad-range exciter filter (360-730) with a 420 
barrier filter (we use the U-MNU cube on an Olympus BX50 microscope), as one 
might use for DAPI detection.  This has the major advantage of revealing a 
much stronger GFP signal.  One drawback, however, is  relatively rapid 
photobleaching, so scan quickly.

3.  When a leaf or tissue with many positive cells is identified, the tissue 
is then prepared for serious viewing.  We find that mounting a piece of 
tissue in water and slapping a coverslip on top is unsatisfactory.  The 
intercellular airspaces and air bubbles generated over stomata by live tissue 
causes viewing problems.  To get tissue suitable for viewing and 
photomicroscopy, vacuum-infiltrate the tissue with water by using a side-arm 
flask and suction pump.  Arabidopsis leaves are easy to infiltrate, so one or 
two brief vacuum/release cycles should do the trick.  Mount the tissue in 
water on a clean slide with coverslip.  Make sure that the coverslip is flat 
and that excess water is drawn off the slide...try to have the thinnest film 
of water between the coverslip and tissue without air pockets forming.

4.  Again, quickly scan at 10x with the broad-range exciter filter set as 
above.  When cells are identified for further viewing, quickly switch over to 
the FITC filter set to reduce photobleaching.  At higher magnifications, the 
intensity of GFP fluorescence will seem to increase. 

Other possibly helpful hints include using a high-level expression vector and 
viewing tissue at the periphery of the blast zone.  And for the enthusiasts, 
try confocal imaging with bombarded cells.  Hope this helps.

Jim

James C. Carrington
Department of Biology
Texas A&M University
College Station, TX  77843
Phone: 409-845-2325
Fax: 409-845-2891
e-mail: carrington at bio.tamu.edu



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