RE: [Histonet] frozen tissue

From:"Jo Dee Fish"

Dear Patsy,
Your cryoprotectant solution sounds suspiciously like the solution we use to
store vibratomed sections at -20C to actually keep them FROM freezing.  It
allows us to keep them very cold but without the damage of actually freezing
them.  The only difference is that we add glycerine to the solution.  I
suspect the ethylene glycol is the problem, I don't think it will allow the
tissue to freeze.  Try leaving it out of the mix and just make a 30%
sucrose/0.1M phosphate buffered solution.  Just my thoughts.
Take care,
Jo Dee 

Jo Dee Fish
Research Technologist III
Gladstone Institute of Cardiovascular Disease

Telephone: (415) 734-2567
Fax: (415) 355-0824

Mailing address:  
The J. David Gladstone Institutes
1650 Owens Street
San Francisco, CA 94158 

-----Original Message-----
[] On Behalf Of Patsy Ruegg
Sent: Friday, January 12, 2007 12:54 PM
To: 'histonet'
Subject: [Histonet] frozen tissue

I am having trouble getting tissue to cut that are prepared as such:

"Animals were heparinized through the inferior vena cava under Avertin
anesthesia.  Hearts were removed, cannulated through the aorta, and reverse
perfused with cardioplegia solution (physiological buffer containing high
concentrations of KCl and EGTA) for 5 min at 2 ml/min to clear blood and
fully relax the heart.  Perfusion was then switched to 4% paraformaldehyde
in PBS for 5 min.  Hearts were removed from the perfusion apparatus,
ventricles were dissected free of aorta and connective tissue, and hearts
were stored overnight at 4C in cryoprotectant solution (0.1 M phosphate
buffer, 30% sucrose, 30% ethylene glycol)."

I took these tissues and snap froze them in liquid nitrogen in cryomolds
filled with OCT as I usually do frozen tissue, as I tried to cut them in the
cryostat they seemed raw as if the glycol had effected the freezing, the OCT
surrounding the tissue was well frozen but the tissue seemed not so well

Does anyone have experience with freezing tissues that have been place in
ethylene glycol cryo protectant, I have not seen this before.

Any advice would be appreciated.  Some of these tissues are still in 30%
sucrose which I transferred them to to try and rinse out the glycol.





Patsy Ruegg, HT(ASCP)QIHC

IHCtech, LLC

12635 Montview Blvd. Ste.215

Aurora, Colorado 80045

Phone: 720-859-4060

Fax: 720-859-4110 


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