GFP in fixed tissue?

John Runions cjr41 at cam.ac.uk
Wed Nov 7 04:24:35 EST 2001


Hi Julin,

I have had some success lately at fixing mgfp5er in enhancer trap seedlings
by a fairly standard protocol:

2.5% formaldehyde (i.e. a 15x dilution of formalin or 37% formaldehyde
solution) in Sorensen's phosphate buffer at 4 degrees celsius.  Do this a
few hours to overnight and then wash and store the seedlings in water.

This fixation preserves the gfp fluorescence nicely but tends to add a bit
of formaldehyde autofluorescence.  We use confocal microscopy to examine
these seedlings and I find that the autofluorescence fades very quickly and
leaves the gfp which is still very robust under the laser beam.  It is
subsequent processing steps that prove problematic.  GFP goes away quickly
when treated with anything other than the lowest concentrations of ethanol
or glycerol.  Please if anyone knows of a good way to make these tissues
transparent while retaining the GFP fluorescence, let us know (I'll buy you
a pint!).

Cheers, John.

Julin Maloof wrote:

>  Can anyone recommend a fixation procedure for Arabidopsis seedlings that
>  preserves GFP fluorescence?
>
>  Thanks!
>
>  Julin
>
>  Julin N. Maloof
>  Plant Biology Laboratory
>  The Salk Institute for Biological Studies
>  P.O. Box 85800
>  San Diego, CA 92186-5800
>  (858)-453-4100, x1105
>  (858)-558-6379 (fax)
>  jmaloof at ems.salk.edu
>
>  ---

--
C. John Runions, Ph. D.
Department of Plant Sciences
University of Cambridge
Downing St.
Cambridge
UK   CB2 3EA

email: runions at plantsci.cam.ac.uk
phone: (01223) 766 545

http://www.plantsci.cam.ac.uk/Haseloff/JohnRunions/Home.html


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