X-Message-Number: 25990
From: "Basie" <>
Subject: "swiss cheese effect"
Date: Fri, 8 Apr 2005 23:44:55 -0400

 Interesting advise.


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."

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