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

Wed Jun 15 16:34:37 CDT 2005

Hi Donna, you're welcome. For the hearts, why not perfuse at RT, put in fridge
for 5-10+ min to cool whole heart before embedding in cold OCT and snap
freezing. The main trick is to minimize the temperature difference between the
tissue + OCT and the actual freezing point. If you can get the center of the
whole "block" cooler, then the snap freeze runs quicker. Chilled blocks can be
larger since there isn't so much "thermal distance" to the center. Undiluted OCT
doesn't adhere to tissues if it is colder than about 4C (refrig), so there are
practical limits to how cold you can make the OCT if you apply *after* freezing.
But if you apply the OCT *before chilling*, there should be no limits.
Especially if your tissue can stand in OCT for a few minutes while chilling.
Chill then freeze. 

Isopentane: I typically do what Frieda Carson does and let the isopentane freeze
solid. Then I remove from the LN2 and break it up into a slurry as it warms up
in the metal beaker. I find that a liquidy-slush does a better job extracting
heat. Be sure to agitate the block in the isopentane to keep it in contact with
the colder "ice-cubes". If you don't agitate then the isopentane local to the
block warms up, even though elsewhere it is still good and cold. I freeze muscle
bxs up to 2x2x4cm (that's centimeters) this way with almost no ice artifacts
even in the dead center.

Lastly, if blocks crack, the cracks can be repaired with cold OCT. I find it
very hard to judge when to remove the tissue from the isopentane slurry -- you
can't *see* when the center is frozen. Contrary to what others say, I find it
much better to "over freeze" and deal with the cracks that happen. That's
microtomy. I get much more consistant freeze quality this way. If you pull too
early then ice artifacts will be forming. Uncracked blocks are easier to cut,
but the morphology often suffers.

Thanks for the paper ref -- I do some work on mouse eyes occasionally.

-brice

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-----Original Message-----
From: Donna Harclerode [mailto:dharclerode <@t> macropore.com]
Sent: Wednesday, June 15, 2005 4:42 PM
To: Due, Brice
Subject: RE: OCT... RE: [Histonet] Deeper molds for mouse brains was
cryomolds

Hi Brice

I do not do brains anymore, but the cold OCT idea may work for the
hearts I am freezing in blocks that sometime I get artifact in the
muscle.  The whole hearts with OCT perfused freeze pretty good mostly.
The problem with chilling the OCT for those would be that it would be
too thick to perfuse. (but will check it out- I already dilute the OCT)
I will play with that for future hearts.  Muscle is one of the most
difficult tissues I know so if things work for you they should work for
pretty much anything.  There is a paper in an old JOH where they mix OCT
and aqua mount half and half for retina of eyes.  Kent Keyser was a PI I
worked for years ago that figured that out for more holding power for
delicate retina.  

Thanks for the new ideas!

Donna

-----Original Message-----
From: Due, Brice [mailto:BDUE <@t> PARTNERS.ORG] 
Sent: Wednesday, June 15, 2005 12:45 PM
To: Donna Harclerode; histonet <@t> lists.utsouthwestern.edu
Subject: OCT... RE: [Histonet] Deeper molds for mouse brains was
cryomolds

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

"THE INFORMATION TRANSMITTED IN THIS EMAIL IS INTENDED ONLY FOR THE
PERSON OR
ENTITY TO WHICH IT IS ADDRESSED AND MAY CONTAIN CONFIDENTIAL AND/OR
PRIVILEGED
MATERIAL.  ANY REVIEW, RETRANSMISSION, DISSEMINATION OR OTHER USE OF OR
TAKING
OF ANY ACTION IN RELIANCE UPON, THIS INFORMATION BY PERSONS OR ENTITIES
OTHER
THAN THE INTENDED RECIPIENT IS PROHIBITED.  IF YOU RECEIVE THIS EMAIL IN
ERROR,
PLEASE CONTACT THE SENDER AND DELETE THE MATERIAL FROM ANY COMPUTER."
HIPPA Policy, 2003,  Brigham & Women's Hospital, Boston, MA

-----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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