[Histonet] frozen tissue

[Patsy Ruegg]

I have no way to omit the ethylene glycol as these samples have already been
exposed to it, I am trying to find out if I can recover these samples for
frozen sectioning.  The best I could think of was to rinse them in buffer
and reinfiltrate in 30% sucrose hoping to wash out the glycol and rehydrate
them so that they will freeze and I can get sections, I will let everyone
know how this came out, I am not optimistic about the outcome.
Patsy

-----Original Message-----
From: Geoff McAuliffe [mailto:mcauliff <@t> umdnj.edu] 
Sent: Friday, January 12, 2007 3:05 PM
To: Patsy Ruegg
Cc: 'histonet'
Subject: Re: [Histonet] frozen tissue

Ethylene glycol is the main component of automotive anti-freeze. I would 
omit it from the cryoprotectant.

Geoff

Patsy Ruegg wrote:

>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
>frozen.
>
>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.
>
>Thanks,
>
>Patsy
>
> 
>
> 
>
>Patsy Ruegg, HT(ASCP)QIHC
>
>IHCtech, LLC
>
>12635 Montview Blvd. Ste.215
>
>Aurora, Colorado 80045
>
>Phone: 720-859-4060
>
>Fax: 720-859-4110
>
>pruegg <@t> ihctech.net
>
>www.ihctech.net 
>
>www.ihcrg.org 
>
> 
>
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>


-- 
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
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