On Fri, 17 Dec 1999, David Sanan wrote:

> Can anyone help with this problem?   We routinely fix mouse brains for
> immunohistochemistry and in situ work.  Brains are removed from mice
> very fast , divided sagitally into hemibrains and fixed in 3%
> paraformaldehyde for up to 5 hours.

   This isn't long enough, though you may need minimal fixation.
   It depends what you want to do with the sections.

> Then embedding in OCT and freezing
> in molds happens.  We sometimes, not always, find that
> the centers of the sections are not as well preserved as the marginal
> zones.   Is this a fixative delivery problem or a cryoprotection
> problem?

    You have not done a cryoprotection. (OCT is just physical
    support around the outside of the specimen. You need to
    soak the fixed brain in sucrose (15 to 30%; the stronger
    the better) until it sinks (e.g. 24 h at 4C).

> Should we further subdivide before fixation?  Any
> recommendations.

    Unfixed mouse brain is easily damaged by handling, so the
    less cutting up the better. You didn't say how you did the
    freezing. Unless you have very good cryoprotection, it
    needs to occur very quickly.

>   We have ruled out perfusion fixation because delivery
> of fixative is too slow and mostly via capillaries which clog very
> easily.

    That's not true at all. It takes about 10 minutes to
    perfuse an anaesthetized mouse with 5-10 ml of saline
    followed by about 25 ml of buffered formaldehyde, and
    the capillaries certainly don't clog! This is the fastest
    and best way to deliver fixative to cells in the brain.
    Many research workers say it's the only acceptable way.

>  We can get the fixative to the brain tissue faster by rapid
> removal of brain, division and immersion.  Under 4 minutes.

    The middle of an immersed specimen doesn't start getting
    fixed for some hours, and the chemical reactions of
    fixation by formaldehyde are slow - much more so than
    any other fixative.

> Brain perfusion takes ages and I am not convinced that some
> areas ever "see" fixative.

    This isn't so if the perfusion is done properly. Mice are
    much more of a fiddle than rats, because of their smaller
    size, so it's easy to poke a hole in the heart and not
    have much saline (or fixative) going anywhere. If the
    liver goes pale, that's a sign of a good perfusion.

    So the ideal way is perfuse - dissect - (immerse in
    fixative too, unless contra-indicated) - cryoprotect
    - freeze - cut.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1