Re: freeze fracturing skin samples
The simplest way is to process the samples through to the final
change in 100% EtOH, as for paraffin embedding. At this point, throw
the samples dripping with EtOH into liquid nitrogen. Since there is
no water in the samples, and since EtOH freezes vitreously, there is
no freezing damage. Tightly grip a razor blade with a hemostat, put
the edge of the blade on the skin when you wish to fracture and
either press down or give the lock of the hemostat a sharp rap with a
weighty rod (or the like). You will likely get many pieces, not just
Move the pieces to 100% EtOH, then process as usual.
The sample can also be placed in a "teabag" for processing -- I
forget their official name, but they've been discussed on histonet
before. But. The teabag will also fracture, making gathering up the
pieces more entertaining.
Sometimes the sample has to be immersed in EtOH, not just soaking
wet. In this case, make a pouch out of parafilm (cut out a rectangle
just more than 2X the size of the sample, fold in half, crimp 3 edges
tightly closed, fill with sample and 100% EtOH, crimp closed the 4th
side) and throw this in the liquid nitrogen.
Ethanol cryofracture is discussed in EM texts and in the primary
literature. Sorry, but I don't have any references to hand, just my
quondum memory. A good source of of microscopy tips -- mostly EM, but
still useful to light microscopists -- is this site from the Univ. of
Tips & Tricks of Microscopy
I've been asked to learn how to freeze fracture skin samples. HELP!
Anybody out there know how to do this and can send me a protocol?
thanks a bunch,
Denise Long Woodward
Harvard Skin Disease Research Center
Brigham & Women's Hospital
Supervisor, BBPIC microscopy facility
Department of Animal Sciences
University of Wisconsin
1675 Observatory Drive
Madison, WI 53706 - 1284
voice: (608) 263-4162
fax: (608) 262-5157 (dept. fax)
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