[Histonet] embedding multiple snap frozen brains

From:"David Finkelstein"

Dear Stephen Peters  and Sarah Pixley,
I am very interested in your methods on freezing brains.
My question is I have never been able to cut brains that have been covered
with OCT.  How do you do it.???  Suggestions please?

Most histologists I know leave the bit of brain that they want sticking out
of the OCT.   The OCT is a different hardness to the brain. If cutting
nonfixed brain at -14oc  the OCT is too soft and fixed brains at -25o C the
OCT is to hard.   It is even harder to get the hardness\temperature right
for foetal tissue.
The solution has been leave it sticking out of the OCT.

Assoc. Professor David Finkelstein,
The Mental Health Research Institute of Victoria,
155 Oak Street, Parkville, Victoria 3052 

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Today's Topics:

   1. embedding multiple snap frozen brains for cryosectioning
      (Stephen Peters M.D.)
   2. RE: embedding multiple brains (Pixley, Sarah (pixleysk))
   3. Very long reply about EDTA  RE: [Histonet] rat myelofibrosis
      questions (Gayle Callis)
   4. Help with Fix-all (SAFETY TRAINORLAB)
   5. Old Vibratome parts? (nienhuis@ucla.edu)


Message: 1
Date: Fri, 3 Nov 2006 10:05:49 -0800 (PST)
From: "Stephen Peters M.D." 
Subject: [Histonet] embedding multiple snap frozen brains for
To: Histonet@lists.utsouthwestern.edu
Message-ID: <20061103180549.6051.qmail@web30402.mail.mud.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Dear Zsuzsa,
  You can do embed multilpe pieced in a single plane with ease using my
embedding well bars.  You can wet the snap frozen tissue with a touch of
embedding medium and freeze the pieces into place flat on the well floor. If
you use cold embedding medium t
o complete the block you will not thaw your snap frozen tissue.
   Please visit http://pathologyinnovations.com/index.html to learn more
about my Precision Cryoembedding S`ystem.

Stephen Peters M.D. 
Vice Chairman of Pathology
Hackensack University Medical Center 
201 996 4836
Pathology Innovations, LLC 
410 Old Mill Lane, 
Wyckoff, NJ 07481 
201 847 7600 


Message: 2
Date: Fri, 3 Nov 2006 13:49:10 -0500
From: "Pixley, Sarah \(pixleysk\)" 
Subject: [Histonet] RE: embedding multiple brains
Content-Type: text/plain;	charset="us-ascii"

Dear Zsuzsa:

We cryosection brains also and this way might work for your needs. We
cut the unfrozen brains (after sucrose) down into sections that include
the area we are interested. We make the most posterior slice as close as
possible to a true vertical plane, so that when we set the brains down
on that, they will be flat and we will be cutting as close as possible
to true coronal sections. Then we lay the brain down flat in a plastic
weigh boat (Fisher Sci. has medium and large ones that might hold
several brains at once). Then we fill the weigh boat with OCT compound
and mark the outside edges of the weigh boat with the specimen number,
mark out the dimensions of the brain and put D and V for dorsal/ventral.
Then we sit that weigh boat flat onto a flat piece of dry ice. It takes
about 15 mins for the whole thing to freeze. As it freezes, it moves
down into the dry ice block. When it is done, it is transferred to the
cryostat and we let it equilibrate for at least 15 mins. Sometimes, we
have to wait, so we put the frozen block in the weigh boat into a Ziploc
bag to keep humidity constant and put in the freezer. After freezing, we
also mark the OCT with important numbers and dimensions. When ready to
cut, we pop it out of the weigh boat and use a razor blade to cut down
the outside OCT to a manageable size rectangular block. We use a small
amount of Oct to attach the block to the cryostat chuck and start
sectioning. If the top remained flat during freezing, you can use it to
get the specimen cutting angle correct and you should be set to go once
you get to the tissue. Sometimes we have to fine tune once getting to
the tissue.

I could envision that you could put several mouse brains in one of the
medium or large size weigh boats, or even make home-made boats out of
aluminum foil. However, you will be limited by the size of the cryostat
chuck and by how well you are able to cut such a large block.

Good luck, hope this helps.
Sarah Pixley

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