Thank you for all your replies to my question regarding oil red O.  Here is
my attempt to summarize those replies (and resulting discussions) starting
with the earliest reply.  Please forgive the haphazard manner of
organization.

Jaclynn Lett, Research Assistant     jlett@cid.wustl.edu

Faye and Carl Simons Center for Biology of Hearing and Deafness
Central Institute for the Deaf
4560 Clayton Avenue
St. Louis, MO  63110

voice: 314.977.0257     fax: 314.977-0030
Jaclynn M. Lett, Research Assistant
jlett@cid.wustl.edu

Faye and Carl Simons Center for Biology of Hearing and Deafness
Central Institute for the Deaf
4560 Clayton Avenue
St. Louis, MO  63110

voice:  314.977.0257     fax:  314.977.0030

Ms. Allan-Wojtas,

In response to your request regarding oil red O lipid staining, here are the
replies I received (along with some discussions as well).  I have not had
time to thoroughly digest everything myself, so please forgive the delay
with which I replied and the lenghthy and unorganized manner of compiling
the replies (listed in the order in which they were received).

Jaclynn M. Lett, Research Assistant
jlett@cid.wustl.edu

Faye and Carl Simons Center for Biology of Hearing and Deafness
Central Institute for the Deaf
4560 Clayton Avenue
St. Louis, MO  63110

voice:  314.977.0257     fax:  314.977.0030


----------------------------------------------------------------------------
---------------------------------------
> 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.

After the tissue is embedded, the solvents used (alcohol, propylene oxide)
will have dissolved most or all of the lipids so there will not be much to
stain. If you stain before embedding, dehydration will remove the lipid and
the stain.

> We would also consider tissues embedded in glycol methacrylate.

Forget it if you are using alcohol (or acetone) for dehydration. I don't
know
of any dehydrating agents that will not remove lipids. Perhaps freeze drying
would work, but you still need an embedding media that won't dissolve
lipids.
Even after osmium, some lipid is lost in dehydration.

> We'd like
> to avoid frozen sections because we'd prefer the higher level of detail
> possible with plastic.

So would a lot of us! This is why frozen sections are used for lipid
staining.

Geoff
--
**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029
mcauliff@umdnj.edu
**********************************************
----------------------------------------------------------------------------
--------------------------------
The lipids will be removed by the solvents used in processing.  In tissues
post fixed with osmium tetroxide BEFORE embedding in these plastics, the
osmium will stain lipids black.    

Gayle Callis
MT,HT,HTL(ASCP)
Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
404 994-4303 (FAX)
----------------------------------------------------------------------------
-----------------------------------
The problem with trying to do lipid staining in epon or plastic embedded
tissue is that inorder to embed in epon or gma you usually have to process
in solvents which will dissolve the lipids not to mention that the plastic
itself is a solvent, at least GMA is somewhat.

Patsy Ruegg
----------------------------------------------------------------------------
------------------------------------
I don't know anyone who has been able to "successfully" stain enbloc or
sections from resins with oil red O, Sudan black or any of the other common
lipid stains because the solvents used in the processing are all designed to
remove fats and lipids.  The only way I could get true accurate staining was
with cryo sections.  I have tried so many methods/ resin schedules/ types of
resin and the same problems always occur, if there is enough lipid remaining
to stain, it doesn't give a true representation of location and quantity.
I'm sorry if this doesn't help, other than to perhaps save you some time and
effort.
As always, if you do happen to find someone who swears by their method, I
would love you to email me a copy of it.
Cheers,
Kerrie

Kerrie Holmes
Histology, Microscopy Research
Research Division, Peter MacCallum Cancer Institute
Locked Bag #1 A'Beckett St. East Melbourne 8006
Phone:  9656 1244 / 1242     Fax:  9656 1411
Email:  k.holmes@pmci.unimelb.edu.au 
----------------------------------------------------------------------------
-----------------------------------
I do alot of samples in GMA and have tried oil red O on a couple of
occasions with no luck.  I would be very interested to know if anyone has
done this and using what method.  Good luck.
Regards
Liz

Elizabeth Cox
Fisheries Biologist
Queensland Department of Primary Industries
Northern Fisheries Centre
PO Box 5396
Cairns, Queensland, Australia  4870
Fax: 07 4035 1401
Ph:  07 4035 0158
----------------------------------------------------------------------------
---------------------------------------
	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."
----------------------------------------------------------------------------
------------------------------------
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 
----------------------------------------------------------------------------
--------------------------------------
	Certain stains for light microscopy are not usable if you are trying
to stain for lipid and increase resolution of the sectioned tissue.  One has
to adjust one's thinking and look only at how lipid can be preserved AND
processed into a plastic, which involves solvents such as alcohol during the
dehydration and infiltration steps.  
	If you are an experienced EM person, you have probably noticed that
lipid has been seen as a green color in your semithin secions. If you look
at Toluidine Blue stained semithin sections, lipid remains green, and the
nuclei and cytoplasm are blue.  The resolution of epoxy and the green color
allows for subcellular identification of lipid.

	HOWEVER, the preservation of lipid by osmium can be greatly enhanced
by p-phenylenediamine (use carefully, it can cause contact dermatitis and
asthma).  Osmicate normal time, then start you dehyration procedure 25%,
50%,then put tissue in a 1.0% p-phenylenediamine in 70% ethanol for 15-25
minutes, then finish dehydratation procedure as normal, up to 100% ethanol,
into ethanol/propylene oxide, etc....Semithin (1-4u)sections without any
additional staining will show lipid by light microscopy.  If needed,
counterstain nuclei with methylene blue stain or Toluidine Blue.   

	Glycol methacry. would not work, because none of the lipid will
remain, due to the alcohols used in processing.  Only osmication, and
especially post- osmication treatment of tissue with p-phenylenediamine
perserves and stablizes lipid so it doesn't wash out during dehyration
steps.

Good Luck!

Karen Jensen, M.S.
Associate Scientist and Project Manager
University of Rochester Medical Center
Electron Microscope Research Core
Pathology and Lab. Med.
Rochester, NY 14642 
----------------------------------------------------------------------------
----------------------------------------------
Years ago I prepared a demonstration slide of testes for the Leydig cells
that came from a conventional EM block, Glut/Cacod and Osmium. 1 micron
section stained with Sudan black then Toluidine blue. Beautiful result but
it was only a one-off, although something I'll try again if needed. 
Ian. 

Dr. Ian Montgomery,
West Medical Building,
University of Glasgow,
Glasgow,
G12 8QQ.
Tel: 0141 339 8855.  Extn:6602.
Fax: 0141 330 2923
e-mail: ian.montgomery@bio.gla.ac.uk
----------------------------------------------------------------------------
----------------------------------
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
----------------------------------------------------------------------------
---------------------------------------
Do you have any fixed, unprocessed tissues to work with??  If so, do
frozens on them instead of your blocks. This is what all the ORO protocols
that I have say to do, which makes sense when you consider that all
processing will subject the tissues to lipid solvents, thus all or most of
the lipids will have been removed and plastics are especially good lipid
solvents. 

Connie McManus  
----------------------------------------------------------------------------
----------------------------------------
The block was post fixed with osmium tetroxide/cacodylate then stained with
Sudan black. It was only a wee trial just to see what happened. At the time
I naively thought that the Sudan black must be binding to the osmium fixed
fat. It was only a once of on a single slide. I stained another slide with
Toluidine blue/Pyronine Y and it was just as lovely. Goodness knows what the
answer was. At the time I used to try all sorts of staining combinations on
semi-thin Glut/Osmium resin sections. Some were awful but others showed
promise, unfortunately time didn't permit further investigation. 
        The best staining for Glut/Osmium resin sections, in our hands was
Toluidine Blue/Pyronine Y (Ito & Winchester 1963 J. Cell Biol) and a
Polychrome technique (Pasyk, Bartok & Fabry 1989 Stain Technol 64 (3) 149.
Ian.   

X-Sender: uvsgc@trex2.oscs.montana.edu
X-Mailer: QUALCOMM Windows Eudora Light Version 3.0.6 (32)
Date: Thu, 17 May 2001 09:01:55 -0600
To: "Dr. Ian Montgomery." <ian.montgomery@bio.gla.ac.uk>
From: Gayle Callis <uvsgc@montana.edu>
Subject: Re: Oil Red O staining.

Question:  Was the lipid staining the result of post fixation with osmium
or from Sudan Black?  Or did you try one block not post fixed with osmium,
stained with sudan black and still have same results?  Just curious.

Nice protocol!!
----------------------------------------------------------------------------
-------------------------------
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
----------------------------------------------------------------------------
----------------------------------
I am not sure what precisely you are trying to demonstrate with the oil
red O?

The assumption in the responses I have seen so far is that Sudan black
is staining the same substances as oil red O. This is not necessarily
true and will depend multiple factors. Sudan black B, is known to also
stain phospholipids, not sure that there is any evidence that oil red O
does this. Can use sudan black on paraffin processed sections and see
some of the phospholipid because some is retained during the processing.

If you are trying to stain neutral fats and can accept poorer nuclear
staining then as Ian says use osmium tetroxide as block stain to show
this.
Barry
----------------------------------------------------------------------------
--------------------------------
Osmium tetroxide is also routinely used to fix fat cells so that size can be
determined.  Don't know if this added tidbit helps.
Sylvia Poulos
USDA-ARS-Animal Physiology Research Unit
Athens, GA 30605
----------------------------------------------------------------------------
------------------------------
Lately we have started experiencing atrocious precipitate on our oil red o
slides.  These are frozen sections of skeletal muscle, 8-9 microns thick,
stained with oil red o in propylene glycol method.

These are large, black crystalline deposits (look much like snowflakes) seen
overlaying the entire specimen.  It is happening with homemade oil red o
stain as well as with commercially prepared oil red o from newcomber supply.
We have tried filtering (multiple filtering of the same stain solution),
decreasing incubation times in the stain (3 hours down to half an hour), and
making up new stain with every run.  I am starting to think maybe it is
something funny in the Milwaukee tap water we rinse with that is binding and
causing the problem.  Have not yet tried rinsing slides in distilled water.

Any similar experiences and solutions are appreciated!

Susan Danielson, MS
Neuromuscular Pathology Lab Coordinator
Dept. Neurology, Medical College of Wisconsin
----------------------------------------------------------------------------
-----------------------------
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
----------------------------------------------------------------------------
------------------------------
I, too, was taught that osmium tetroxide reated with the unsaturated lipids
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.
    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
----------------------------------------------------------------------------
------------------------------
> 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)
----------------------------------------------------------------------------
------------------------------------
If you use the Churukian method for Oil REd O, you get NO ppt on your
sections.  It eliminates the need for messy propylene glycol, and is a
clean, gorgeous stain.  Chuck published the method in the last two years, J
of Histotechnology.  

I never looked back once I started to use it.

At 04:29 PM 5/17/01 -0500, you wrote:
>following the thread on oil red o:
>
>Lately we have started experiencing atrocious precipitate on our oil red o
>slides.  These are frozen sections of skeletal muscle, 8-9 microns thick,
>stained with oil red o in propylene glycol method.
>
>These are large, black crystalline deposits (look much like snowflakes)
seen
>overlaying the entire specimen.  It is happening with homemade oil red o
>stain as well as with commercially prepared oil red o from newcomber
supply.
>We have tried filtering (multiple filtering of the same stain solution),
>decreasing incubation times in the stain (3 hours down to half an hour),
and
>making up new stain with every run.  I am starting to think maybe it is
>something funny in the Milwaukee tap water we rinse with that is binding
and
>causing the problem.  Have not yet tried rinsing slides in distilled water.
>
>Any similar experiences and solutions are appreciated!
>
>Susan Danielson, MS
>Neuromuscular Pathology Lab Coordinator
>Dept. Neurology, Medical College of Wisconsin
>
>
Gayle Callis
MT,HT,HTL(ASCP)
Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
404 994-4303 (FAX)
----------------------------------------------------------------------------
------------------------------


	-----Original Message-----
	From:	Paula Allan-Wojtas [SMTP:AllanWojtasP@EM.AGR.CA]
	Sent:	Friday, May 18, 2001 11:03 AM
	To:	jlett@cid.wustl.edu
	Subject:	Re: Oil Red O lipid staining

	Dear Jaclynn,

	I am interested in this, too. Would you mind sharing and replies you
get with me?

	Thanks a lot in advance.

	Paula.

	Paula Allan-Wojtas
	Research Scientist - Food Microstructure
	Agriculture and Agri-Food Canada
	Atlantic Food and Horticulture Research Centre
	Kentville, Nova Scotia Canada B4N 1J5

	Tel: (902) 679-5566
	FAX: (902) 679-2311

	email:   allanwojtasp@em.agr.ca