On Fri, 14 Jul 2000, Bruce Abaloz wrote:

> Dear Histonetters.....my name is Gratiana & I am wanting to process some VERY tiny/fragile & "precious" specimens of Tammar Wallaby Day 12 - 17 vesicles. I am hoping that someone out there has worked on specimens this fragile/minute & can kindly email me their processing schedule/procedure...one that I have a copy of from our Histologist , "layers" gradients of alcohol eg.30,50,70,90,100% in a measuring cylinder & says to put the specimen @ the top layer...BUT the 100% alcohol always goes to the top & so on until the 30% is bottom!? I have tried processing as a pellet in agar but that needs to be fine tuned & as specimens are @ this time hard to come by. I do not want to take chances. Please - if you have a suggestion I will be MOST APPRECIATIVE & THANKYOU in advance.

  One method is to put the delicate specimen (must be adequately fixed,
  of course) in 10% glycerol, then leave it in an open jar with suction
  (e.g. a vacuum desiccator, or a vacuum oven that's not switched on)
  until the water has evaporated - volume down to 10%; mark the level
  on the side of the jar when you start. Alcohol can then be added
  gradually to the glycerol + specimen until it's about 95% alcohol
  and 5% glycerol. Then go into 100% alcohol. (You have to go into
  alcohol eventually, because glycerol doesn't mix with clearing
  agents or wax.)  I've done this occasionally, and it takes a couple 
  of weeks to evaporate the water with our building's rather feeble 
  vacuum line. End results are fine. 

  This glycerol method for dehydrating delicate specimens is often
  used for bits of plant that have complex shapes but consist mostly
  of water. Rapid or even moderate solvent changes will make the
  cellulose cell walls collapse. For a detailed account of the method,
  Berlyn,GP & Miksche,JP (1976) Botanical Microtechnique and
  Cytochemistry. Iowa State Univ Press, Ames. This book, which you
  can buy directly from the publisher, is a nicely bound
  hardback, is full of useful information and is unbelievably cheap
  (oops! - inexpensive).

  Rather surprisingly, chemical dehydration with DMP is also quite 
  gentle on delicate specimens. (It was first used for scanning EM.) 
  It's described in Berlyl & Miksche (a pretty new method back in
  1976) and there's a more recent account in Biotechn. & Histochem.
  74: 20-26 (Jan 1999). The DMP method is much faster than glycerol,
  needing only 10-15 minutes for a 1 mm specimen, so you might want
  to try it first.  

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