[Histonet] frozen tissue

[James Watson]

Propylene glycol or Ethylene glycol is used in cryoprotectants for brain
when the thick sections are cut on a sliding microtome.  It keeps the
tissue from freezing completely and the section is lifted off the knife
with a paint brush then floated onto a slide.  This method can produce
extremely nice thick section.  If you need the exact protocol I would
have to dig it out of my files.

James Watson HT, ASCP
Facilities Manager of Histology
GNF, Genomics Institute of the Novartis Research Foundation
Room C015
858-332-4647
jwatson <@t> gnf.org  

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Geoff
McAuliffe
Sent: Friday, January 12, 2007 2:05 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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>  
>


-- 
--
**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 
mcauliff <@t> umdnj.edu
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