7/22/96
We are interested in embedding a variety of connective tissue matrices, most
from tissues including skin and cartilage, in OCT in preparation for cryostat
sectioning. Our protocol in the past has been to freeze in "hexanes" cooled to
just above its freezing point of -94 C in liquid nitrogen, store in hexanes
at -70C, then embed in OCT and cool to -20 C prior to cryostat sectioning. We
are concerned that there may be a freezing protocol resulting in more favorable
structure. We are concerned that the freeze-thaw-freeze occuring as the tissue
is frozen, then placed in OCT at ambient temp., then frozen again may not be
optimum, even for LM.
Recently, we have considered the use of other freezing media, including
isopentane. We've noticed that some other laboratories are embedding fresh
tissue in OCT, then freezing the entire block in isopentane prior to cryostat
sectioning.
As we make our choice in deciding how to proceed, perhaps others with
experience in freezing tissue in preparation for LM immunocytochemistry might
contribute opinions based on their experience. Our goal is for adequate
stabilization, long term storage of unembeddedd tissue, and good adherence of
tissue to OCT.
Many Thanks,
Doug Keenee
DRK@shcc.org
the University of Wash, in Dr Holbrooks lab.
We routinely place fresh skin samples directly into OCT as soon as
possible and freeze them in either a isopentane or ethanol and dry ice
slush. Isopentane works better. The ethanol can make the OCT turn to
rubber if it comes in contact with it. The blocks are stored in -70
freezer and some have been cut periodicly for 10 years. When using
desposable knives, however, the tissue can detach from surrounding OCT. I
haven't found anything better easier yet. But if you can cool the
isopentane in LN2 it is better but sometimes unavailable.
Bob
underwoo@u.washington.edu
We routinely freeze brain tissue in our EM/Imaging lab. We take fixed
tissue and run it through a sucrose series (10%, 20%, and 30%) to
cryoprotect. We then place the tissue in OCT in a foil cup and place that
on dry ice cooled with acetone. We then store that at -20'c until we
section.
If you would like more details, let me know. We are having pretty good
success.
Cheri Owen
Detroit Neurotrauma Institute
Detroit, Mi 48201
313-285-4027
I have been freezing skin equivalents made of collagen I and III in OCT
(Miles) using isopentane for some time and have found storage of tissue
to be the major obstacle to good histology. If you know someone with
some cryobottle space or a cryofreezer try to store there. Typically my
preps have good overall structure up to 3-4 months at -80, and now about
a year in storage in a cryofreezer.
If you have many different stains to run on the tissue you may want
to section twice as many slides as you think you might need, fix in
methanol or acetone for 10 min. at 4 C and then store the slides at -70
to -80 C. I found this method easier than freezing and thawing my
original tissue each time I wanted to stain for something.
Hope it Helps,
Steve Hendrix
s002swh@desire.wright.edu
Rogosin Institute
Ohio Branch