Re: Plastic Embedded Nerve. Best way to fix.

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From:"J. A. Kiernan" <> (by way of histonet)
To:histonet <>
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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
    card index.)
  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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