"Dr. Ian Montgomery." <ian.montgomery@bio.gla.ac.uk>
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<x-tab> </x-tab>Just been
reading the posting by Phil and the answer by John. Slam freezing,
couldn't be easier and all with home made equipment. <br>
EQUIPMENT:<br>
<x-tab> </x-tab>Solid
copper rod, 2x3 inches. Get your workshop to polish one end. Then sit
with fine paper and polish to a near mirror finish and finish with metal
polish. A friendly EM Unit is handy, they usually have polish for the
Wehnelt cylinders. Then attach the polished block to an insulated rod or
if you can find one, a racket that you can wind up and down.<br>
<x-tab> </x-tab>Bamboo
sticks, barbecue type. Buy a pair of Sorbothane insoles then with a punch
cut out pieces with a similar diameter to the bamboo sticks. Glue
the Sorbothane cores to the bamboo sticks.<br>
<x-tab> </x-tab>Millipore
filters, or similar, cut into small pieces.<br>
METHOD:<br>
<x-tab> </x-tab>1.) Fill a
wide mouth dewer, almost to the top, with LN2.<br>
<x-tab> </x-tab>2.) Place
the copper rod into the dewer and leave to cool. Topping with LN2 if
necessary.<br>
<x-tab> </x-tab>3.)
Dissect out the tissue and cut into small pieces under a suitable Ringer
solution.<br>
<x-tab> </x-tab>4.)
<b>Quickly, </b>place a piece of tissue onto a moistened filter then on
top of the Sorbothane.<br>
<x-tab> </x-tab>5.)
<b>Even more quickly,</b> raise the copper block <b>just out</b> of the
nitrogen, then with one even movement slam the tissue against the
polished surfaced.<br>
<x-tab> </x-tab>6.)
Transfer to a pot of LN2 and cut of the Sorbothane core. <br>
<x-tab> </x-tab>The frozen
tissue can now be sectioned or freeze substituted and embedded for
sectioning.<br>
NOTES:<br>
<x-tab> </x-tab>1.) Keep
the level of LN2 high thus avoiding pre-cooling and freezing.<br>
<x-tab> </x-tab>2.) Only
bring the copper block just out of the LN2 and only before you are going
to slam. Any longer than necessary and frosting will occur, a real
bummer. <b>You must have a polished surface.<br>
<br>
</b><x-tab> </x-tab>I've
used this technique and published the results for many years. Once you
perfect the skill it's really easy. Using this technique my lab has
produced some stunning electron micrographs from freeze substituted
tissue. I've even got one on the wall at the back of my computer, now is
that not sad.<br>
Ian. <br>
<x-tab> </x-tab>
<br>
<x-sigsep><p></x-sigsep>
<font color="#0000FF">Dr. Ian Montgomery,<br>
West Medical Building,<br>
University of Glasgow,<br>
Glasgow,<br>
G12 8QQ.<br>
Tel: 0141 339 8855. Extn:6602.<br>
Fax: 0141 330 2923<br>
e-mail: ian.montgomery@bio.gla.ac.uk</font></html>