X-Message-Number: 25990 From: "Basie" <> Subject: "swiss cheese effect" Date: Fri, 8 Apr 2005 23:44:55 -0400 Interesting advise. Basie This is a quote! " I am sectioning fresh rat spinal cords on our cryostat at -20C, and after histochemical tests the slides show lots of holes. Is there a way to ensure that holes do not develop on the sections? You also mentioned that you are dipping them in isopemtane to keep them cold and freezing them in OCT.. Holes in the tissue, called "swiss cheese effect" are a well known phenomena in histology of brain. They are also called freezing artifact, caused by large ice crystals forming and rupturing cell membranes. There are many ways to control them. Another possible cause of something like this is autolysis caused by delayed fixation (you are using fresh tissue, fixation is freezing the tissue). The time from when the tissue might be expected to become anoxic, until it is fully frozen, must be minimized. Rapidly frozen tissue (3 or 4 seconds to solid) has small crystals that do not cause freezing aritifact. This requires immersion in -80 C or colder fluid, or complete embedding in powdered CO2. Cold (-80 C) isopentane is great for full immersion of the tissue. Howeve, if after being so frozen, the tissue sits around at -20 for a long period, overnight, say, small crystals will reform and become large crystals, and you will be back to freezing artifact, even though you initially froze fast. Examine your procedure. Freeze in morning by full immersion in -80 C isopentane. Place the tissue in the cryostat to warm up to cutting temperature. Begin sectioning as early as possible without getting shatter artifact. Finish that day, or re-store at -80 C. Freeze the tissue by immersion as soon as you can, even piling dry ice over it while it is still in the animal, then extracting it. One frozen, put a little refrigerated (4 C) OCT on a refrigerated pedastal, then drop the tissue on and dip the pedastal to quick freeze the OCT. You probably do not need to fully embed in OCT. Let me know if this helps." Copyright 2001 Vibratome.com Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=25990