Re: 10% NBF for TEM fixation?

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From:"J. A. Kiernan" <> (by way of Marvin Hanna)
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On Wed, 19 Jan 2000, Carla Aiwohi wrote:

> Recently we received whole #003#zebrafish mistakenly fixed in 10% NBF.
>We are
> interested in examining muscle with TEM and usually fix tissues in modified
> Karnovsky's then post-fix in 1% osmium tetroxide.  Would it be better to
> fix directly from NBF or should I re-fix (?) in Karnovsky's and carry on as
> usual?

   I would recommend post-fixing in a glutaraldehyde-containing mixture
   (Karnovsky's would be OK) after the neutral formalin and before the
   osmication. This will introduce many more covalent cross-links than
   you could ever get with formaldehyde alone, and the electron
   micrographs will, with luck, resemble the ones you get with primary
   fixation in Karnovsky's mixture. Do you use the original Karnovsky
   (1965) brew, with 4% glutaraldehyde? For many years most who fix
   for EM have used only 1-2.5% glutaraldehyde. The concentration is
   not critical. For more information, see M.A. Hayat's Principles and
   Techniques of Electron Microscopy, where this sort of stuff is
   thoroughly discussed.

Also, why isn't 10% NBF suggested as a primary fixative for EM?

   It is, and it does a pretty good job if given a week or two to
   do its stuff. In about 1968 I saw some excellent EM pictures of
   human thyroid from a colleague who collected archived
   leftovers from surgical specimens. See also the usual textbooks,
   especially Hayat's mentioned above. One NBF variant developed
   especially for EM is that of Carson, Martin & Lynn, 1973 (Amer.
   J. Clin. Pathol. 59: 365-373). It is mentioned quite frequently
   in HistoNet questions and answers, often as Carson's fixative and
   is, of course, also a perfectly good neutral formaldehyde fixative
   for light microscopy. For what it's worth, my favourite neutral
   formaldehyde for LM is Lillie's formal-calcium acetate, mainly
   because it needs almost no work to make it up, and the Ca ions
   make it lipid-friendly. I haven't tried it as a primary fixative
   for EM, and haven't seen anything in print either. Probably
   someone has tried it. A search of PubMed would probably come up
   with something.

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

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