On Fri, 20 Oct 2000, Rebecca S Smith wrote:

> ANYWAY!  My real question for you Histo Gurus out there is 
> this:  Why?  What is the purpose?  Did someone get this crazy idea because 
> they had some Cedar Wood Oil in the back closet (for use on the family 
> heirloom) and it worked and now it's gospel on how to do this Carnoy's 
> processing?

  It isn't gospel at all. Most people take tissue from Carnoy to
  100% alcohol, then clear in xylene or similar.

  Here is a bit of scanned text from the 3rd (1974) edn of Culling's
  Handbook (p.80). I think there's a similar piece in the more recent
  edition (Culling, Russ Allison & Barr, 1985) but I don't have that
  book to hand. 

  "Cedar-wood oil is the best reagent for research and treatment of
delicate tissue, since it has the least hardening effect.  Tissue may be
left in this clearing agent for long periods, even for months, without 
damage.In some institutions it is used as a clearing agent for hard tissue
such as skin, and dense fibrous tissue, since sections are easier to cut 
after such treatment.  When ordering it should be specified that the
cedar-wood oil is for use as a clearing agent.
  "Even very small pieces of tissue need to be left overnight in
cedar­wood oil; specimens from 5 to 7 mm in thickness will need 2-5 days.
Cedar-wood oil may be poured into a small specimen jar, and a similar
quantity of absolute alcohol superimposed on it, avoiding any mixing at
the -junction, of the two fluids: the specimen is then placed gently into
the alcohol when it will float at the interface of the two fluids.  As
clearing takes place the specimen slowly sinks into the cedar­wood
oil; the alcohol is then removed by pipette or syphon, the specimen
transferred to fresh cedar-wood oil for a few hours, and finally
transferred to paraffin wax.
  "Following clearing in this reagent, several extra changes of paraffin
wax will be required to remove the oil. This technique is rarely, if ever,
used today.

"Technique of Clearing

 "The technique of clearing is the same as that used in dehydration,
tissues being lightly blotted during transfer from one reagent to the next.
The volume of clearing agent should be 50-100 times that of the tissue.
Tissues being cleared in chloroform or carbon tetrachloride are best left
overnight, and those in xylene, benzene or toluene should be given one
change after 30-60 minutes, and transferred to wax when they are seen to
be clear (translucent).  The special technique for cedar-wood oil has been
dealt with above."

  Another book that has quite a lot to say in favour of cedarwood oil is
  M. Gabe's "Histological Techniques" (1976). He said it facilitated the
  preservation of delicate organelles such as mitochondria and their
  subsequent staining by dyes in paraffin sections. I don't know any
  textbook that says you've got to use cedarwood oil after Carnoy, and
  the "delicate organelle" argument is inappropriate because this fixative
  destroys them all. It's very good for nuclei, cytoplasmic RNA and mast
  cell granules, and the tissue architecture as seen with lower
  magnifications is well preserved. Paraffin sections seem to cut more
  easily than after formalin (perhaps because of more complete
  infiltration; this is a general thing with non-aqueous fixatives).

  Don't fix in Carnoy for longer than overnight. 4-6 hours is good enough
  for small (2-3 mm) objects. Excessive exposure to the acetic acid in
  the fixative can extract RNA. I once had a series of mouse brains that
  lost all their Nissl substance after being left 24 hrs in Carnoy, so
  it's really true. There is a variant with only half as much (5%)
  acetic acid, specially recommended for when you want to keep as much
  RNA as possible in the sections (James J & Tas J 1984. Histochemical
  Protein Staining Methods. Oxford Univ Press. It's #4 of the
  Royal Microscopical Societies excellent little paperback handbooks;
  probably out of print by now.)

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