RE: [Histonet] frozen tissue

From:"Surita Banwait"

Hi There,
Yes we also use this solution to 'cryoprotect' (keep tissue from
freezing/forming ice crystals) vibratome free floating sections.

When running IHC on these samples , we give the tissue thorough washes
with PBS 

Surita Banwait
Morphology & Imaging Core
Research Associate II         
Buck Institute for Age Research
8001 Redwood Blvd.
Novato, CA 94945

-----Original Message-----
[] On Behalf Of Jo Dee
Sent: Friday, January 12, 2007 1:14 PM
To: 'Patsy Ruegg'; 'histonet'
Subject: RE: [Histonet] frozen tissue

Dear Patsy,
Your cryoprotectant solution sounds suspiciously like the solution we
use to
store vibratomed sections at -20C to actually keep them FROM freezing.
allows us to keep them very cold but without the damage of actually
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
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
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
perfused with cardioplegia solution (physiological buffer containing
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%
in PBS for 5 min.  Hearts were removed from the perfusion apparatus,
ventricles were dissected free of aorta and connective tissue, and
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
cryostat they seemed raw as if the glycol had effected the freezing, the
surrounding the tissue was well frozen but the tissue seemed not so well

Does anyone have experience with freezing tissues that have been place
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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