On Tue, 14 Dec 1999, Emma Carter wrote:

> > Would you mind telling me your timings for hand processing......!?

SLOW METHOD.

  For pieces 3 to 5 mm thick, in 25-30 ml vials (polyethylene
  scintillation vials with screw caps are cheap & strong).
    70% alcohol 2 hours (or overnight)
    95%         2 hours
   100%         2 hours
   100%         2 hours
     The clearing agent (next 2 steps) is terpineol (my favourite)
     or benzyl benzoate or cedarwood oil. The used stuff from the
     2nd step is kept and re-used once for the first step for
     later specimens.
   Clearing agent  2 to 24 hours
   Clearing agent  2 to 24 hours
   Benzene, toluene or xylene   15-30 minutes
     (This is to remove excess of the oily,
      non-volatile clearing agent from the
      surface of the specimen.)
   Wax:  4 changes, each 1 hour, but often longer,
     in a heated container connected to the building's
     "vacuum" line.  This is where the pieces
     sometimes get forgotten for a day or two!

------------------------------------

FAST METHOD.

  For most purposes these days we do chemical
  dehydration in acid-catalysed 2,2-dimethoxypropane
  (DMP) instead of using alcohols. It is cheaper to use
  than alcohol (because only one change is needed),
  and also less trouble. DMP reacts with water,
  the products of the reaction being  methanol and
  acetone, which are miscible with all (or most)
  clearing agents, as is DMP itself. (Cedarwood
  oil is not fully miscible with methanol, so is
  perhaps better avoided, or preceded by toluene or
  xylene; haven't tried this.)

   Add one drop (0.01 ml) of conc. hydrochloric
   acid to 50 ml of 2,2-dimethoxypropane (DMP).
   Use 5 to 10 ml for each specimen (assuming
   5 mm cubes). Leave for 30-60 minutes, with
   an occasional shake. Then transfer the
   specimen to any clearing agent.

   Recently we have found that this method, once
   believed suitable only for small objects, can
   fully dehydrate 2 cm cubes of tissue if left
   overnight. For more details, and a discussion
   of the economics, see Biotech. Histochem.
   74: 20-26 (1999).
------------------------------------

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