Re: freezing artifact in perfused, frozen mouse brain

From:Stephanie Moore <>

Hi Stephanie,

I will agree with everyone about your problem:  Definitely the
cryoprotection with only 10% sucrose.

The idea of using graded sucrose concentrations has to do with the
osmotic difference between your tissue and the sucrose solution (makes
sense and is what I have been told, anyway).  If you went straight into
30% sucrose, the difference is so great that the water would be "sucked"
(in effect) out of the tissue (most evident in cortex, so if that is
what you look at, as I do, you want to do everything possible to keep
that tissue normal).  Going from 10%-20%-30% (until sinking each time)
is better since it is "gentler" on the tissue.  

If the tissue is sufficiently cryoprotected, then freezing it on dry ice
(I assume that you place the chuck onto the dry ice and then put OCT on
it, place the specimen and finish covering with OCT?) is okay and you
should not have freeze artifacts. I do rat and squirrel brains in this
way with no problem.  If you don't use higher than 10% sucrose, then you
need to speed up the freezing process so that the ice crystals are
minimal if not nonexistent.  Most people snap freeze their specimens in
isopentane cooled in either liquid nitrogen or in dry ice (look in
archives for the how-to's).  I have just started doing this and it is
great (saves time, too).  Freezing that fast means that cryoprotecting
is not so crucial.

I know this is basically what you got in the other replies...I just
thought you might want to know some "whys"...

Good Luck,
Stephanie Moore
Brandeis University
Biology Dept, MS8
Waltham, MA  02454  

Stephanie Rodriguez wrote:
> Hello!
> First, I would like to thank everyone who responded to my question regarding
> paraffin-embedded brain coming apart on the water bath.  I got lots of
> suggestions, and upon further review, my tissue was definitely underfixed; I
> fixed another set for 24hrs in 10%NBF at RT, and got MUCH better results.
> With the other suggestions, such as turning down the water bath, soaking,
> etc, I was even able to get a few sections out of the underfixed brains.
> Thanks!!
> Now, I have a new problem.  A research tech in our lab is perfusing mice and
> freezing the brains.  He has done this procedure in the past, and is using
> the same protocol he used then, but we are getting alot of freezing artifact
> in the cortex.  Could you please evaluate his protocol to see if you can
> help us solve this problem?
> Here it is:
> 1.  Anesthetize mouse
> 2.  Open chest cavity and cannulize left ventricle.
> 3.  Run ~20mL saline, then cut right atrium.
> 4.  Run ~200mL 4% paraformaldehyde in Na Borate buffer, pH 9.5.  (Why
> borate?  I don't know.)
> 5.  Remove brain and place in a jar with the above fixation solution, and
> fix O/N @ 4C.
> 6.  Place in 10% sucrose (* I was always told it to use 30%, but he insists
> 10% works fine) and
>         allow to sink (also @4C).
> 7.  Freeze on dry ice and store until ready to cut on cryostat.  (My job)
> When I cut these tissues, they seem really fragile, even at 20u, and when I
> lay them on a slide, they wrinkle up.  Under the microscope, the ice crystal
> artifact is blazingly obvious.
> What are we doing wrong?   Clearly, they are not adequately cryoprotected
> when frozen.  Are they insufficiently fixed?  Do we need more sucrose?  I
> have a protocol I got from the Histonet a few weeks back that used
> polyethylene glycol as well as sucrose in the cryoprotection step.  Should
> we try that?
> Help!
> Stephanie Rodriguez, B.S., HTL(ASCP)
> Research Associate III/Histotechnologist
> Primal, Inc.

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