Fwd: Re: Slam freezing.

From:"Dr. Ian Montgomery." <ian.montgomery@bio.gla.ac.uk>

<html> <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>
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