Re: Plastic Embedded Nerve. Best way to fix.
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|From:||"J. A. Kiernan" <firstname.lastname@example.org> (by way of histonet)|
On Mon, 5 Oct 1998, Catherine Johnson wrote:
> We've had a problem with our plastic embedded nerve biopsies on and off.
> The tissue sections are stained with MAB or Toluidine blue. Often times,
> it looks like there has been poor penetration of the fixative or possibly
> other solutions during processing. Sometimes the sections are difficult to
> read because there is so much artifact. Artifacts include poor penetration
> of the stain, distorted cross sections of myelinated fibers, and wrinkles.
> Our EM technician has a tendency to always blame it on the fixative, so she
> makes an effort to make sure the fixative is always fresh. We've also
> started slitting the perineurium with a razor blade before dropping the
> specimen into fixative in hopes of improving penetration, ....
You don't say where the specimens of nerve are from. If they are
from laboratory animals, the following fixation method is even
better than vascular perfusion. It might be adapted for biopsies.
Anaesthetize the animal and expose the part of the nerve to be
fixed. While looking at the nerve through a dissecting microscope,
manipulate the limb (or other part of the body) to stretch the
nerve until its transverse striations (bands of Fontana) just
disappear. (The bands of Fontana are due to undulations (waves)
of the nerve fibres - a bit of microanatomy that was cleverly
deduced before 1700, long before the fibres themselves had been
properly seen.) Having straightened the nerve's fibres in this
way, drip the fixative (a buffered glutaraldehyde & formaldehyde
mixture) onto the exposed nerve. Apply a pellet of cotton wool
soaked in the fixative, and wait for 30-60 minutes. Remove
the desired specimen and immerse it in fixative for a further
6-12 hours. Wash in buffer, post-osmicate, embed, section,
stain (if desired), etc.
The superiority of this method over simple immersion or perfusion
was demonstrated by Morris et al (1972), in one of four classical
papers on the ultrastructure of the earliest stages of axonal
regeneration. For the story of the bands of Fontana (a conspicuous
but rarely discussed feature of nerve anatomy), see Hanicek 1986
(and also papers in J. Anat. by Bearn & others, 1970s, which I
can't find just now). My second graduate student, Bruce
Stelmack, was able to obtain useful measurements of external
(with myelin) and external (axon) nerve fiber diameters in
4 micron paraffin sections of facial and sciatic nerves of rats
that had been fixed by the method of Morris et al.
Hanicek 1986. Anatomy & Embryology 174 407-411. (Sorry, this is
the best I can do from home, with just the computer and not the
Morris,JH; Hudson,AR; Weddell,G (1972): A study of degeneration and
regeneration in the divided rat sciatic nerve based on electron
microscopy. IV. Changes in fascicular microtopography, perineurium and
endoneurial fibroblasts. Zeitschrift fur Zellforschung 124, 165-203.
(This is THE classic in its field. Weddell was the boss. His work in
the early 1940s with J. Z. Young and W. Holmes provided the
scientific foundation of modern peripheral nerve repair surgery.)
Stelmack, B. M. & Kiernan, J. A. 1977. Effects of triiodothyronine
on the normal and regenerating facial nerve of the rat.
Acta Neuropathologica 40, 151-155.
(Bruce Stelmack's sciatic nerve study was published only as an
unrefereed abstract, and in his MSc thesis, because the results
conflicted curiously with some earlier ones from our lab. Later
work may have reconciled the conflict.
If, O individual HistoNetter, you have an interest in the
currently unfashionable matter ofthyroid hormones and axon
regeneration, please get in touch, and let's exchange some
thoughts and references!) "Little does he know that I know ...
End of long, boring aside." (N. Seagoon)
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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