I support Tom's answer - for fixing tissue in situ for subsequent TEM,
we routinely perfuse the animal with fixative. But we have found that it
makes a huge difference to the overall "quality" of the fixation (note
Tom's caveats!) if we pre-perfuse with fully constituted tissue culture
solution first (to wash out all the blood). If you do this with saline
or Ringer's or anything less than a culture medium, the tissue looks
really bad - full of holes and lots of components go missing...

In case you may be thinking this is getting off the confocal path, we
have found time and time again that if we want to get the best cellular
/ subcellular preservation for light microscopy following experiments in
vitro (eg recording from neurons in isolated tissue), then the more
"complete" the medium, the better everything is. If you have a look TEM
at a prep that has been all day in some sort of balanced salt solution,
you don't see much more than a few bags of membranes (which is just
aabout all you need for electrical recordings...but it does make you
wonder!)

If people want more info on fixation chemistry, protocols etc, look up
any of the excellent books by Hayat or Glauert.

Hope that helps

IAN



Tom Phillips wrote:
>
> Good question.  My answer is NO.  The big question is do you count
> the osmolarity of the aldehyde fixatives?  Their ability to penetrate
> tissue and cross membranes has been questioned but most of the
> molecules must not contribute since the osmolarity of a 10% solution
> of formaldehyde is 1300 mOsm and of a 4% solution is 520 mOsm.
> Adding glutaraldehyde increases it even further.  On the other hand,
> the fix I use has salts (NaCl, HEPES, CaCl2) that is hypo-osmotic (I
> forget but I think it is around 225 mOsm instead of physiological of
> 300-308) and yet my tissue looks excellent (in my humble
> opinion.....).  I forget what the osmolarity of salts in Karnovsky's
> fix is but I think I modeled my concentrations on his.  But it could
> be what I (we) think is excellent fixation is artifactual.  The
> typical text book views of brain tissue shows little or no
> extracellular space between all the neurons, glia, etc.  But Van
> Harreveld showed that quick-frozen brain tissue had lots of space.
> The classic view of mitochondria with cristae and open space is a
> result of chemical fixation - in quick-frozen tissues, it is
> difficult to see cristae since the mitos are so condensed.  If you
> add ox phos uncouplers prior to quick-freezing, the mitos look
> "normal".  Some think fixation osmolarity is of little consequence
> since fixation is "fast" but there have been nice studies of
> electrophysiological recordings of miniature end plate potentials at
> neuromuscular junctions showing the electrical signals changing over
> a time course of minutes during fixation.  The bottom line is we
> usually end up using what gives us a pretty picture but we should
> always remember, it might not be a perfect reality.  My thesis
> adviser always called it chemical "embalming" rather than fixation.
>
> In an earlier posting of a similar question,
> *************************************************************** Geoff
> McAuliffe, Ph.D.
> Neuroscience and Cell Biology
> Robert Wood Johnson Medical School
> 675 Hoes Lane Piscataway, NJ 08854
> voice: (908)-235-4583; fax -4029 e-mail: [log in to unmask]
> ************************************************************* said:
>
> "In the theme of Diana van Driel's post check out Arborgh et al. "The
> osmotic effect of glutaraldehyde during fixation". J. Ultrastr. Res.
> 56:339-350, 1976. A very interesting and through study".
>
> I haven't gotten around to looking this article up yet.  Good luck.  Tom
>
> >Are there generally accepted rules for selecting osmolarity of fixative
> >solutions for TEM?
> >Thanks group!
> >
> >Milton Charlton
> >University of Toronto
> >
> >-----
>
> --
> Thomas E. Phillips, Ph.D.
> Associate Professor of Biological Sciences
> Director, Molecular Cytology Core Facility
>
> 3 Tucker Hall
> Division of Biological Sciences
> University of Missouri
> Columbia, MO 65211-7400
> (573)-882-4712 (voice)
> (573)-882-0123 (fax)

--
Professor Ian Gibbins
Anatomy & Histology
Flinders University of South Australia
GPO Box 2100, Adelaide, SA 5001
Australia

Phone:  +61-8-8204 5271
FAX:    +61-8-8277 0085
Email:  [log in to unmask]