[Histonet] frozen tissue

nienhuis <@t> ucla.edu nienhuis <@t> ucla.edu
Fri Jan 12 18:13:33 CST 2007


I would appreciate a copy of the exact protocol. Something similar has
been suggested as a way of preserving antigenicity of formalin fixed
stored tissue.

How thick are these thick sections?

Bob Nienhuis
UCLA / VA Medical Center

Quoting James Watson <jwatson <@t> gnf.org>:

> 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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