John:
We have had no problem imaging fluoresence from EGFP vector in rat or mouse
bone using our standard formalin fixed, formic acid decalcified paraffin
embedded protocol.I cut the sections at 4 microns, placed them on regular
slides, deparaffinized in three changes of xylene, applied No 1 coverglass,
and imaged them on our Zeiss CSLM at 488nm excitation. The fluorescence is
relatively permanent. I've put the slides back on the scope after a month
(the investigator wanted to show off to someone else) and the fluorescence
was just as bright. If you have specific questions respond back channel.
(we're in the process of publication). Donna Montague, UAMS Orthopaedic
Research

-----Original Message-----
From: bakerj@umich.edu [mailto:bakerj@umich.edu]
Sent: Wednesday, June 09, 1999 3:02 AM
To: Histonet@pathology.swmed.edu
Subject: Rat GFP Tissues


Histonetters:

    I have some whole Rat bone (femur) specimens that I expect to be
expressing GFP.  I want to look for GFP in transverse (circular)
cross-sections of the femur.  A colleague had suggested that I use Spurr's
Resin to embed the entire bone and then image the cross-sections using
confocal.  This sounds like a great idea to me, however, since GFP is
quickly degraded with alcohol there are major hurdles to overcome.

I am faced with the following questions regarding these tissues:   After
fixing the femurs in 4% paraformaldehyde, what non-alcohol solution should
I store them in until I embed them?  In the protocol for embedding in
Spurr's I obviously need to dehydrate the tissues;  how does one do that
without alcohol?  Is there a different method someone might know of to
image transverse cross-sections of femur and still be able to image GFP
fluorescence?

Thanks in advance for your assistance!

John



John A. Baker
The University of Michigan
Orthopaedic Research Laboratories
Histology Unit
400 North Ingalls, G161
Ann Arbor, MI 48109-0486
my office 734-936-1635
lab office 734-763-9674