On Sun, 20 May 2001, Dr. Allen A. Smith wrote:

> in the cell membrane.  I cannot see how this can be true.  The lipids are
> the middle layer of the membrane.  If osmium tetroxide reacted with the
> unsaturated lipids, we would see the cell membrane as a single dark gray
> line in electron micrographs.  Since we see the membrane as two black lines,
> osmium tetroxide must be reacting with the amino alcohols which form the two
> outside layers of the membrane.

This was a controversy in the early days of electron microscopy, and is
discussed in textbooks (Hayat, Glauert etc). It's some time since I read
about this stuff, but OsO4 is certainly reduced to OsO2 by alcohol
groups and also by some amino acid side-chains. However, it's much more
soluble in hydrophobic domains (middle of membrane) than in hydrophilic
ones (inside or outside surface). The unit membrane (Robertson)
appearance was explained at least partly by migration of the insoluble
OsO2 molecules (or of an osmate indermediate) from the hydrophobic middle
of the membrane to the more hydrophilic inner and outer surfaces. Osmium
reduction by protein was also recognized, as was extraction of cytoplasmic
and other proteins into primary OsO4 fixatives.

>  Osmium tetroxide reacts with fat in fresh tissue by reacting with the
> double bonds in oleic acid.    If one cuts thin sections for electron
> microscopy, fat droplets are gray because there isn't that much oleic acid
> present.  If one cuts thick sections, one sees fat droplets as black because
> many layers of gray look black.
> 
> Allen A. Smith, Ph.D.
> School of Graduate Medical Sciences
>    Podiatric Medicine and Surgery
> Barry University
> Miami Shores, Florida
> 
> ----- Original Message -----
> From: Morken, Tim <tim9@cdc.gov>
> To: <Histonet@Pathology.swmed.edu>
> Sent: Thursday, May 17, 2001 1:05 PM
> Subject: RE: Re Oil red staining
> 
> 
> > Well, for whatever reason osmium will preserve large fat droplets in
> tissue
> > and I have used it to demonstrate lipid storage diseases in paraffin
> > sections. So, I can't give you the chemistry, just the observed results.
> >
> > Tim
> >
> > -----Original Message-----
> > From: Philip Oshel [mailto:peoshel@facstaff.wisc.edu]
> > Sent: Thursday, May 17, 2001 11:21 AM
> > To: Morken, Tim
> > Cc: Histonet@Pathology.swmed.edu
> > Subject: RE: Re Oil red staining
> >
> >
> > A question: is this really the case? Osmium binds across double-bonds
> > of lipids, which is why it shows and preserves membranes, but it
> > binds poorly or not all at to saturated lipids. So I wouldn't expect
> > OsO4 to show fatty deposits if the fats are saturated. Oil Red O and
> > Sudan Black, which more mix into the lipids and don't react with
> > them, would show fat deposits that OsO4 doesn't, and are better fat
> > stains.
> >
> > Phil
> >
> > >Agreed, there would be no need to do special fat stains if the tissue is
> > >processed with osmium. In fact, this can be done for paraffin embedding
> as
> > >well to show fat distribtion in some diseases with vastly better
> morphology
> > >and localization than frozen sections.
> > >
> > >Tim Morken
> > >CDC, Atlanta
> > >
> > >-----Original Message-----
> > >From: Saby, Joseph [mailto:Joseph.Saby@pfizer.com]
> > >Sent: Thursday, May 17, 2001 6:15 AM
> > >To: 'RichardWHorobin@aol.com'; Histonet@pathology.swmed.edu
> > >Subject: RE: Re Oil red staining
> > >
> > >
> > >Richard-
> > >
> > >Whenever I've worked with those plastics, there has always been a
> clearing
> > >stage through acetone.  Since acetone would remove all non-bound fat, Oil
> > >Red O would have nothing to go into.
> > >
> > >When I've worked with these plastics, I also did a post-fixation in
> osmium
> > >tetroxide, which does a very good job of staining fats and lipid (it
> turns
> > >them black).  Perhaps this would work for your purposes.
> > >
> > >Joe
> > >
> > >Joseph A. Saby, BA, HT(ASCP)
> > >Drug Safety Evaluation
> > >Pfizer Global Research and Development
> > >2800 Plymouth Road
> > >Ann Arbor, MI 48105
> > >Phone: (734)-622-3631
> > >FAX:   (734)-622-3866
> > >E-mail: joseph.saby@pfizer.com
> > >
> > >
> > >
> > >-----Original Message-----
> > >From: RichardWHorobin@aol.com [mailto:RichardWHorobin@aol.com]
> > >Sent: Thursday, May 17, 2001 2:49 AM
> > >To: Histonet@pathology.swmed.edu
> > >Subject: Re Oil red staining
> > >
> > >
> > >Jaclynn Lett
> > >wrote:
> > >
> > >
> > >
> > >Has anyone used Oil Red O to stain lipids in tissues embedded in plastics
> > >(Epon or Epon-Araldite)?  If so, has this been done by staining en bloc
> or
> > >by staining the sections.  Sections would range from 1-4 microns in
> > >thickness.
> > >
> > >
> > >
> > >A couple of people so far have commented on how hard this would be, and
> our
> > >experience agrees with theirs.
> > >HOWEVER Jaclynn also said:
> > >
> > >
> > >
> > >
> > >We would also consider tissues embedded in glycol methacrylate.  We'd
> like
> > >to avoid frozen sections because we'd prefer the higher level of detail
> > >possible with plastic.
> > >
> > >
> > >
> > >In THAT case things look better! Would you settle for Sudan black, rather
> > >than Oil red staining of the lipid? If 'yes' then there is a method -
> which
> > >does indeed show even tiny droplets of lipid very clearly. This was
> worked
> > >out by the one-time king of GMA staining Peter Gerrits, and can be found
> in
> > >J
> > >Neurosci Methods, as follows:
> > >
> > >      Gerrits PO, Brekelmans-Bartels M, Mast L, 's-GrAavenmade EJ,
> Horobin
> > RW
> > >
> > >and Holstege G. (1992)..
> > >      Staining myelin and myelin-like degradation products in the spinal
> > >cords of chronic experimental
> > >      allergic encephalomyelitis (Cr-EAE) rats using Sudan Black B
> staining
> > >of glycol methacrylate-embedded
> > >      material.
> > >      J. Neuroscience Methods. 45, 99-105
> > >
> > >Bye - Richard Horobin
> > >
> > >Institute of Biomedical & Life Sciences, University of Glasgow
> > >T direct 01796-474 480 --- E  RichardWHorobin@aol.com
> > >"What should we expect? Everything."
> >
> > --
> > }}}}}}}}}}}}}}}}}{{{{{{{{{{{{{{{{{
> > Philip Oshel
> > Supervisor, AMFSC and BBPIC microscopy facilities
> > Department of Animal Sciences
> > University of Wisconsin
> > 1675 Observatory Drive
> > Madison,  WI  53706 - 1284
> > voice: (608) 263-4162
> > fax: (608) 262-5157 (dept. fax)
> >
> >
> 
> 
>