[Histonet] frozen tissue

Maria Mejia mbmphoto <@t> gmail.com
Sat Jan 13 12:57:57 CST 2007


In our lab, we lots (LOTS) of large (3X2.5 inche) primate brain  
blocks & rat whole brains
Here's what we do to get beautiful 40um cryosections. The primate  
brain blocks are:
1. fixed.
2. washed X2 in working PBS to remove residual fixative and then
3. placed in cryoprotectant solution of PBS & 30 % sucrose until the  
tissue sinks to
the bottom of container @ 4C. This takes several days.
4. tissue blocks are blotted using kimwipes to remove excess sucrose  
(before) freezing using
the liquid nitrogen & metal block method. We use this method of  
freezing primate blocks because we
absolutely required that our tissue have a flat cutting surface.
5. after freezing the blocks are wrapped twice w/foil & placed in  
freezer zip-loc bag w/silca gel and
packed into cryo boxes and stored at -80C freezer - to be cut later  
on the cryostat.

Now for mice or rat brains:
1. after cryoprotectant solution, again whole brains are blotted  
using kimwipes to remove excess sucrose
and then frozen on a cryo chuck using cryo-gel (we don't use OCT -  
too running). Beside cryo-gel holds
much better and keeps tissue colder.
2. during sectioning for both primate & rat brain, we use anti-freeze  
solution in culture plate well to place
serial sections. The anti-freeze solution has ethylene glycol/ 
glycerin (99%)/PBS.
3. we use a sable brush to wet the knife edge w/anti-freeze solution.  
As a section is cut, it slides on top
of the knife w/anti-freeze solution. Just simply use the brush to  
lift the section off the knife & place into
a well with more anti-freeze solution.
**after learning this technique - it's fast, easy to section tissue  
and not as messy as you might think.
4. the plate wells full of cut sections in anti-freeze solution are  
sealed with cryo-tape and stored at 4C
temp in plastic boxes.
5. we do IHC/IFA on these sections including H&E and Nissl staining -  
even on those sections stored for
several months.

**Now, if you need to store the sections for long term in anti-freeze  
solution at 4C - add a few grains of thymol
into the solution as a preservative.

I hope this helps.

Maria Bartola Mejia
Department of Neurosurgery
San Francisco, CA

On Jan 12, 2007, at 2:29 PM, James Watson wrote:

> 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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