POLYMER DISKS FOR FLAT EMBEDDING<br>
________<br>
This is particularly suited to resins that must be polymerized in the absence of oxygen, but
works well for any resin.<br>
<p>  Flat embedding can be difficult with the acrylic resins
since the polymerization is oxygen sensitive.  Elaborate
devices to exclude oxygen have been reported.  We have
taken to using a paper punch to make circles of EM film
(unexposed but cleared with fixer) or Aclar membrane
(Allied Chemical, Pella) which turn out to be a size that will
go partway down a 1.5 ml polypropylene Eppendorf tube
thus forming a platform on which a piece of tissue might
rest.  The film can be coated with poly-L-lysine which will
help cells from suspension adhere.  Aclar film can also be
used to grow cell cultures so that they might be embedded
in situ (Kinglsley, 1988).  After polymerization of the resin
the block is removed from the tube.  The block will separate
into two parts at the film since neither of these materials
will embed.  This leaves the tissue or cells at the surface of
the cleavage ready to section in any orientation and is the
equivalent of flat embedding (suggested by the work of
Ridgeway & Chestnut, 1984).  The technique can be used
with either epoxies or acrylics, however the polyester film
backing of EM film is not as effective in epoxy resins as it is
in acrylic resins.  (See figure below.)<br>
<p>
Kingsley, R. E. and N.L. Cole  1988  Preparation of cultured
mammalian cells for transmission and scanning electron
microscopy using aclar film.  J. EM Tech  10:77-85.<br>
<p>
Ridgeway, R. and M. Chestnut  1984  Processing small
tissue specimens in acrylic resins for ultramicrotomy:
improved handling and orientation.  J. Electron Microscopy
Tech.  1: 205-206.<br>
<p>
<center>
<img src="disk.gif">
</center>
<p>
<ul>
<li>A. A film disk  is placed in the tube to be used as a platform for previously fixed.
dehydrated and infiltrated tissue or:<br>

<li>B. Cells grown on an Aclar disk are fixed, dehydrated and infiltrated before placing in
a tube.  Or fixed cells may be deposited on a film disk charged with poly-l-lysine<br>
(The disk is  difficult to keep level, so we sometimes precast some epoxy resin in the
bottom of the tube to a level that will provide support for the disk.  Aclar film tends to
curve a bit, so if it is important to keep it planar, one can "spot weld" it to an EM film disk
with a hot dissecting needle before use , to provide dimensional stability.)<br>
<li>C. Tubes are filled with resin. making sure no air bubbles remain.  (Not necessary if
oxygen is OK)<br>

<li>D. Tubes are closed and polymerized by the chosen method.<br>

<li>E. The tube containing the polymerized plastic is sawn down the side and the
specimen removed.<br>

<li>F. The resin will cleave at the plastic disk with a little encouragement.<br>

<li>G. The specimen is exposed at the surface of the block, giving immediate access for
sectioning.<br>

FROM G. Erdos (gwe@biotech.ufl.edu)<br><br>
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