[HISTONET] fixation and storage of tissue for TEM

[John Kiernan]

Overnight in glutaraldehyde is more than adequate for the tiny (<1mm) bits of tissue that are needed for transmission electron microscopy. Two hours is often sufficient. The fixation, washing, postosmication, propylene oxide (if used) and preliminary infiltration with catalyzed epoxy resin can then be done within one working day. Polymerization in a capsule at 60C is then an overnight step. 
 
If glutaraldehyde-fixed specimens must be held for more than an hour or two, it should be in buffer or water. At least one animal tissue (CNS) does not respond to changes in osmotic pressure after glutaraldehyde fixation (Paljarvi, Garcia & Kalimo 1979 The efficiency of aldehyde fixation for electron microscopy: stabilization of rat brain tissue to withstand osmotic stress.  Histochemical Journal 11:267-276).  I hope some histonettters will reply to the group with information less than 30 years old about liquid storage of other fixed tissues for later TEM. 
 
There are some very good books that provide instructions for preparing tissues for electron microscopy. I highly recommend an inexpensive paperback:
Hunter, EE (1993). Practical Electron Microscopy. A Beginner's Illustrated Guide, 2nd ed. Cambridge: Cambridge University Press. ($30 new; $7 second-hand, on Amazon.com.)
Some histotechnology textbooks have brief but good good EM chapters. An example is: 
Brown GG (1978). An Introduction to Histotechnology. New York: Appleton-Century-Crofts. (On Amazon.com this book is priced over $200 but ordinary second-hand bookshops have it for $5 to $10!)
 
Every lab that does TEM should have access to a library that contains the big books in this field: notably the several volumes edited by Glauert and Hayat. The preparative techniques for TEM were understood and perfected before 1970, so it isn't necessary to have the latest editions.
 
John Kiernan 
Anatomy, UWO
London, Canada
= = = 
----- Original Message -----
From: Nicholas David Evans <ndevans <@t> stanford.edu>
Date: Tuesday, December 8, 2009 16:58
Subject: [HISTONET] fixation and storage of tissue for TEM
To: histonet <@t> lists.utsouthwestern.edu

> Dear all,
> 
>  
> 
> I would like to fix and preserve mouse skin tissue for 
> processing for
> transmission electron microscopy (TEM) at a later date. I was 
> wonderingwhether I can store tissue following fixation overnight 
> in 2.5%
> gluteraldehyde in 0.1M sodium cacodylate buffer and, if so, 
> whether it can
> be stored indefinitely in this buffer (or whether I need to 
> treat with
> OsO4 and store at 70% ethanol)?
> 
>  
> 
> Thanks and best wishes
> 
> Nick
> 
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