freezing artifact in perfused, frozen mouse brain

From:Stephanie Rodriguez <>


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.

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?


Stephanie Rodriguez, B.S., HTL(ASCP)
Research Associate III/Histotechnologist
Primal, Inc.

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