Re: freezing artifact in perfused, frozen mouse brain

From:"A. Erickson" <>

First, he may be using borate buffer at that ph to preserve/detect some
neurotransmitters in the brain.  The fixation sounds adequate, but what is
the sucrose made up in?  Maybe wash out the fix with 0.1M phosphate
buffer, sink in graded concentrations of sucrose in 0.1M phosphate buffer
(10-20-30%) freeze on dry ice, cut using cryostat at colder temperature
than for fresh(unfixed)brain.  Also, he may be used to working with
free-floating sections, cut using a sliding microtome, where the 10%
sucrose was sufficient.  If he says this works, maybe he should try
cutting the sections himself?  Just a thought.  Andra Erickson, UW RPRC
Neurohistology lab, Seattle, Wa.

On Tue, 19 Jun 2001, 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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