RE: GFP in paraffin

From:"Montague, Donna C"

I have used anti-GFP antibody from Clontech to compare the localization
against the EGFP (enhanced stability). I found that the EGFP fluorescent
signal is stable to alcohol thus standard paraffin processing protocols work
well. The advantage of the antibody is that the observer can choose the
detection method for their color preference and perhaps perform
dual-labeling techniques for light microscopy. This approach may be
desirable for your application as apototic cells can be labelled with TUNEL
and the the GFP can be immunocomplexed and detected with NBT/BCIP or other
chromogen. Should make quite the pretty picture. I have also used the GFP
primers to amplify the incorporated construct in situ and then detect the
amplimer via immunochemical methods. I can send you my protocols for Insitu
PCR if you think that would be helpful.

Kindest regards, Donna Montague, M.S.
Research Associate
Physiology/Biophysics and Orthopaedic Surgery
University of Arkansas for Medical Sciences
(501) 603-1239

-----Original Message-----
From: Tom Clarke []
Sent: Monday, March 18, 2002 11:11 AM
Subject: GFP in paraffin

Hi Dr. Montague,

   I've heard through the histological grapevine that you are a bit of
an expert at visualizing GFP in sectioned tissues.  Do you have any
experience in using anti-GFP antibodies to detect enhanced GFP in
paraformaldehyde fixed paraffin embedded sections?  I'm attempting to
visualize eGFP in apoptotic cells and having problems with the low
levels of eGFP expression that build up in the tissues dissappearing in
the embedded sections (non-apoptotic cells with high levels of
expression still show GFP after sectioning) and am thinking of using
anti-GFP antibodies to 'find' the lost GFP.  Do you know if the GFP
protein is being lost from the cells (and thus undetectable to anti-GFP
antibodies) or whether it stays in the cells and is merely denatured to
an extent that it no longer fluoresces?

  -Tom Clarke-
  Division of Biology
  Kansas State University

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