On Tue, 10 Nov 1998, PAMELA HORGE wrote:

> out staining to "totally uninterpretable".  We have already increased our
> fixation and the processing times with little improvement. The current
> protocol is as follows:

> Zenkers (with zinc not mercury) minimum 1 hour
   This is a very short time. The Zn and acetic acid will
   have time to do their stuff, but the reaction of tissue
   components with dichromate is much slower (though not as
   slow as formaldehyde). Why not fix overnight?

> 100% ethyl alcohol 8 min at 67 degrees

   After fixing in Zenker or any other dichromate-containing
   mixture, you need a long wash in lots of water before
   moving into any kind of alcohol. A 1 cm cube needs
   overnight in running tap water. For a tiny biopsy the
   time is doubtless shorter. To test the wash, leave the
   specimen in a tiny volume of water for an hour. If any
   yellow (dichromate) comes out into the water, washing
   is inadequate.
     If you move a specimen that contains dichromate into
   alcohol, the two substances react (oxidation & reduction)
   and an insoluble green chromium(III) compound, probably
   Cr(OH)3 or similar, precipitates in the tissue. (With
   Helly's fixative this occurs in a controlled manner
   and is a desired effect, but not with Zenker.)

> Isopropyl alcohol 8 min at 74 degrees
> Paraffin 3 min at 64 degrees, same container reset for 5 min at 82
> degrees

  Isopropyl alcohol does not mix with paraffin, and at
  82 C it will boil within the specimen, so it's not
  surprising that you see damage! Surprisingly, it is
  possible to get way with going from isopropanol
  into wax, but the conditions (temperature and
  pressure) have to be exactly right or there will
  be all sorts of artifacts. See Bosch MMC, Walspaap,CH
  & Boon,ME 1996. Eur. J. Morphol. 34: 127-130.
    Even though it's from the place that developed
  the isopropanol --> wax trick, reading this paper
  should deter most people from trying it.
    If you want to dehydrate specimens quickly
  the easiest reagent is acidified 2,2-dimethoxypropane
  (DMP). This reacts chemically with H2O, generating
  acetone and methanol. A little object like a biopsy
  will be ready to move into a clearing agent after
  15-20 minutes. Chemical dehydration has been around
  for more than 20 years. In our lab we've found
  that it will also dehydrate big specimens if
  given enough time. Because you need only one
  change, it's cheaper than using alcohol (paper
  in press in Biotech. Histochem.).

  To summarize: Wash your Zenker fixed pieces
  _thoroughly_ before dehydrating, and use a
  clearing agent between the alcohol and the
  wax.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1
   Phone: (519) 679-2111
   FAX (Department): (519) 661-3936
   E-mail: kiernan@uwo.ca