On Fri, 19 Nov 1999, Joyce Kotzuk wrote:

> ...they were too long in 100% EtOH/Cedarwood oil (50-50 mix). Although
> the cedarwood oil is protective, the 100% EtOH may still have been
> too drying.

   You can't remove too much water. The wax won't go where
   there's water or alcohol. All the water originally in a
   specimen must be replaced by clearing agent in order to
   allow penetration by the wax. Are you sure your 100%
   alcohol is dry enough? If there is humidity it will
   soon be 98%, which is no good. You can protect your
   100% alcohol by putting molecular sieves in the bottom
   of the bottle to extract water.

>               ... I do 3x15 minutes in 100% EtOH/Cedarwood oil.
> I don't use xylene at all, cedarwood oil and methyl salicylate
> instead, then paraffin. Comments/questions about this are welcome.

   Cedarwood oil is good, but its viscosity slows down its
   penetration through the specimen and its extraction into
   the melted wax. Allow lots of time and several changes of
   wax. You can store specimens for years in cedarwood oil.
   A short treatment with chloroform or xylene after the
   oil and before the first wax may speed up the displacement
   of oil by wax. Some cedarwood oil will remain in the
   tissue despite several long immersions in hot wax, and
   this may well be a good thing (as discussed in my HistoNet
   posting on this topic a few days ago).

   For intelligent discussion of this sort of stuff, sit
   down for an hour or two with "Histological Techniques" by
   Manfred Gabe (Paris: Masson, 1976). The subject is also
   gone into at length by Peter Gray (1954) in "The Microtomist's
   Formulary and Guide." The latter book is less easy to
   read because of the way it's organized. Gabe was a French
   zoologist and an authority on neurosecretion. He did all his
   own lab work without any technical help, and his "Histological
   Techniques" is consequently full of useful maxims. He died
   shortly before the publication of this big fat book.

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