Message: 22
Date: Wed, 14 Jul 2004 09:49:11 +0200
From: DANIEL EBERHARD 
Subject: [Histonet]         related question; but FREEZE- Formalin
fix---then
        freeze?
To: histonet@lists.utsouthwestern.edu
Message-ID: <40F4E577.8090000@UNI-BIELEFELD.DE>
Content-Type: text/plain; format=flowed; charset=ISO-8859-1
 
Dear Histonetters,
I have a succeeding question to the "formalin-freeze" topic.
Accidentally some mouse tissues (with cytosolic and highly soluble GFP),
which I routinely pre-fix in pFA, wash and freeze, were frozen without 
pre-fixation (stored at -800C).
Now, I would like to *thaw *the tissue, *fix it* (pFA) and *freeze* it 
again, but of course thawing (e.g. crystal formation)
might ruin cells and membranes, so that the GFP might pour out. Thus, 
I4m searching for some tips now to manage this?
Has anyone done sth. like this before?
"Post-pFA-fixation" of sections is unfortunately impossible.
 
Thanks for any tips and tricks.
Daniel.

Daniel,
I think I can help you with this one. I think I am getting to the age
that I have made most of the mistakes or had to deal with them. I once
had a bull dog submitted for a post mortem with a threat of litgation
because it died at the groomer. These dogs can die from any kind of
excitement because they have so much redundant pharyngeal tissue to choke
on. The bad part was the dog was quick frozen on dry ice before
submission. It was going to take a couple days to thaw it out. I got
tired of waiting and figured it would be a gross diagnosis anyway so I
posted it when it was crunchy and I could just move the limbs. I put
crunchy tissues in formalin figuring the freeze artifact would be
terrrible in any case. I have gotten tissue in the mail in the winter and
tissue frozen in formalin is the worst. To my surprise the tissues looked
great. Now I don't hesitate to drop frozen tissue into formalin and let
it thaw and fix at the same time.

I also work with GFP but use formalin fixed paraffin embedded tissue and
a MAb against GFP after Dako antigen retrieval in the autoclave. I use
Vector ABC alk phos and Vector red. The results are spectacular bright
red staining and hematox counterstain. I highly recommend it. Forget
using FA. The signal was not that great and it would fade as well by FA.
These slides are permanent. If someone really wants fluorescence, the
Vector red lights up great with a Texas red filter. If you are doing
confocal, just switch the colors to green if you like green. The signal
is even better than the red staining using white light. I have found
weakly stained cells that I could not see any other way. I think it is
the way to go. Good luck.

Doug Gregg
Veterinary pathologist
Plum Island Animal Disease Center

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