Re: freezing artifact in perfused, frozen mouse brain

From:Geoff McAuliffe <mcauliff@UMDNJ.EDU>

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.

Why so much?? Where could one possibly put 20 ml in a mouse's circ.system? Cut
the rt. atrium as soon as the flow starts.

> 4.  Run ~200mL 4% paraformaldehyde in Na Borate buffer, pH 9.5.  (Why
> borate?  I don't know.)

I have used borate, it works OK. Sodium phosphate dibasic by itself is 8.9 or
so. You can add some NaOH to raise the pH if you like.

> 5.  Remove brain and place in a jar with the above fixation solution, and
> fix O/N @ 4C.

Why switch from RT to 4 degrees C?

> 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).

If 10% works fine, why are there ice crystals?

> 7.  Freeze on dry ice and store until ready to cut on cryostat.  (My job)

If only 10% is going to be used, one must freeze very quickly to avoid ice
crystal formation, in my experience. The brain must be surrounded with crushed
dry ice.

> 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?

Not enough cryoprotection and/or freezing too slowly.

> Clearly, they are not adequately cryoprotected
> when frozen.  Are they insufficiently fixed?

Maybe, but with all of the ice crystal holes, who can tell?

> 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?

More cryoprotection AND faster freezing.

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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