OCT... RE: [Histonet] Deeper molds for mouse brains was cryomolds

[Due, Brice]

Hi Donna, I primarily work with human muscle bxs, which are sensitive to
conditions that produce ice artifacts. My cold OCT trick may apply to rodent
brains. I keep a bottle of OCT in a regular 4C refrigerator. I use it to embed
troublesome muscles *after* they have been snap frozen in isopentane. No ice
artifacts show up except maybe the outermost muscle fiber or two -- often none
at all. After applying the cold OCT, I use either a freeze spray or dry ice or a
-80C freezer to finish the job. 

For brains you could try snap freezing specimen and post-embedding like I do
muscles. Or you could try using the cold OCT to fill your molds before freezing.
This moves the specimen + OCT temp 15-20C south of RT, which means the
isopentane has much less work to do to cross the freezing point. I don't freeze
whole OCT blocks often, but I have noticed that cold OCT has fewer cracking
problems than RT OCT when rapidly frozen. 

-brice
Neuropathology Lab
Brigham & Women's Hospital
Boston

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Donna
Harclerode
Sent: Wednesday, June 15, 2005 1:59 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Deeper molds for mouse brains was cryomolds


Hi Betsy

I use PEEL-A-WAY molds made by POLYSCIENCES INC (available from VWR) for
my bigger specimens. I do not use them for brains. The problem with
trying to embed a brain in any sort of orientation in a mold is
something I have never solved.  When I put the brain in OCT it distorts.
Rat and mouse brains cut very nicely (much better for me) if they are
frozen by themselves.  The 2 ways I do it is to make a foil boat and set
the brain in and put it on dry ice with or without isopentane(slabs of
dry ice are much better than pellets because they have a flat surface)
for a minute or 2 (rats are more on the 2 minute side) till the top is
frozen.  The only thing with this is you get a flat spot on the bottom
of the brain. These brains went for RNA ISH and we did 70 a week so we
needed speed. 

The other way that takes a bit more effort is to hold the brain by the
spinal cord and lower it into the isopentane in a beaker in dry ice
10-20 seconds depending on temp. One PI I worked with had a specific
temperature range for the isopentane, but I do not remember that now. If
you freeze it too long, you can get cracks or even multiple pieces if
you leave it in way too long. After the brain is frozen (less is better
than over freezing) 

I put the brains in conical tubes on dry ice till I get them to the -80
freezer. I have not had problems with ice artifact with either of these
methods.  For OCT embedded "regular" tissue either of these 2 methods
have given me ice artifact either due to the warmer temperatures or
increased time because of the OCT. I always use isopentane and liquid
nitrogen for tissue other than brain Maybe the high lipid content in the
brain makes the ice artifact less of a problem.
To cut these I put a bit of OCT on the chuck and put the brain in the
position I wanted. I usually cut the rostral end for coronal sections to
make a flat spot for the chuck. One other tip we did was to have a small
container of OCT and quickly dip the frozen mounted brain and refreeze
the block.  I made a hollow of dry ice in the container, dipped the
brain and refroze the brain as quickly as possible.  

Good luck

Donna Harclerode, HT, (ASCP), HTL, QIHC
Immunohistochemist
MacroPore Biosurgery, Inc
6740 Top Gun St.
San Diego, CA 92121
858-458-0900 xt 322
dharclerode <@t> macropore.com

Subject: RE: [Histonet] cryomolds
To: "

Try some of the molds sold for plastic embedding which are deeper and
then
transfer to your cryostat chucks.  

Shirley 
 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
Molinari,
Betsy
Sent: Wednesday, June 15, 2005 5:48 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] cryomolds

Hi all,
 Does anyone know if there are cryomolds that are deeper than 5mm? I
have
some mouse brains to cut and the researcher wants to keep them intact
and
not bisect them, any suggestions?
Thanks,
Betsy Molinari HT(ASCP)
Texas Heart Institute
Cardiovascular Pathology
Houston, Texas 77030
832-355-6524
832-344-6812 (fax)
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