"Dr. Ian Montgomery." <ian.montgomery@bio.gla.ac.uk>
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<br>
<blockquote type=cite class=cite cite>Date: Tue, 3 Oct 2000 08:53:07
-0500<br>
To: "Dr. Ian Montgomery."
<ian.montgomery@bio.gla.ac.uk><br>
From: Philip Oshel <peoshel@facstaff.wisc.edu><br>
Subject: Re: Slam freezing.<br>
Cc: Histonet@Pathology.swmed.edu<br>
<br>
Ian,<br>
<br>
Thanks! A nice procedure. Slam-freezing can produce some nice results,
and can be the only way to get well-frozen biopsies (with one of the
slam-freezing "guns"). But. The layer of well-frozen tissue is
not much thicker than that obtained by plunge-freezing into slush
nitrogen, and the slamming does create compression artifacts, possibly
even disruption. Have you had the opportunity to compare your slam-frozen
samples to propane-jet or high-pressure frozen
samples?</blockquote><br>
<x-tab> </x-tab>We used
liquid propane (with a wee bit of isopentane) and slamming. Interestingly
the best results for freeze sub came from slamming but for X-ray
microanalysis liquid propane was best. <br>
<br>
<br>
<blockquote type=cite class=cite cite>Rereading your post, are you
slamming the tissue into the copper block by hand? One quick
"HaiYAH!" and whack! ? If so, this isn't the slamming I meant.
I was thinking of the slammer devices that propel the tissue at a rapid
rate into the cooled block. This causes compression artifacts. If you are
doing this by hand, then no matter how fast you are, you're not
"slamming", but rapidly freezing by contact to a cooled metal
block. Similar to plunge freezing, without the Leidenfrost problem, but
not as efficient as plunging into slush nitrogen. You will get -- as your
images show -- excellent freezing of the outer layer of the tissue, but
this won't extend much below 10, maybe 20 microns (some tissues do
better, but not a whole lot).</blockquote><br>
<x-tab> </x-tab>Yes, I
slammed by hand. Quickly raise the copper block then with one swoop slam
the tissue. In our naivety we reckoned that the shock absorbing
properties of the Sorbothane would have a protective activity. Whether it
did or not I couldn't say but, the results were good. We tried without
the Sorbothane, straight onto the stick and with other base materials but
Sorbothane was still best. I agree 20 microns was as good as we got,
after that ice crystals started to appear. <br>
<br>
<br>
<blockquote type=cite class=cite cite>You do do something that is
critical to getting the best morphology from freeze-fixed tissue:
freeze-substitution. Do you have a special recipe for
this?</blockquote><br>
<x-tab> </x-tab>Again, my
freeze sub technique is very simple. No elaborate equipment, all home
made except for the -80 deep freeze. Visitor just arrived will finish
posting later.<br>
Ian.<br>
<x-tab> </x-tab><br>
<br>
<blockquote type=cite class=cite cite>Phil<br>
<br>
<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.)
Quickly, place a piece of tissue onto a moistened filter then on top of
the Sorbothane.<br>
<x-tab> </x-tab>5.) Even
more quickly, raise the copper block just out 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. You must have a polished surface.<br>
<br>
<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. <x-tab> </x-tab><br>
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<br>
<br>
-- <br>
}}}}}}}}}}}}}}}}}{{{{{{{{{{{{{{{{{<br>
Philip Oshel<br>
AMFSC and BBPIC<br>
Dept. of Animal Health and Biomedical Sciences<br>
University of Wisconsin<br>
1656 Linden Drive<br>
Madison, WI 53706-1581<br>
voice: (608) 263-4162<br>
fax: (608) 262-7420 (dept. fax)<br>
</blockquote>
<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>