Mauricio Morales spotted a problem in the first DVD of muscle/nerve
techniques. It has to do with the freezing of the muscle. I show a thin
walled plastic mold with just a little OCT in the bottom of it to hold
the muscle or nerve in the mold during freezing. The next shot shows a
mold with muscle in it more completely covered with frozen OCT.
Mauricio points out that this is confusing.  Where did that added OCT
come from? ----sorry for the confusion. The second shot, with the more
OCT around the tissue, was taped a year or so after the actual freezing
episode. The idea of that second shot was just to show how to bolster
the tissue with OCT, to make a good block for cutting.  The block I used
for that shot did have more OCT frozen around it than it would have had
if it had just been frozen. Sorry about that. The actual sequence is
this:  1.  A small amount of OCT is put in the bottom of a thin walled
mold to keep the tissue in the mold while it is frozen. The tissue is
properly oriented for cutting cross sections. It is frozen.  2:  The
layer of frozen OCT holding the tissue in the mold is thin and quite
fragile,  so a little OCT is added to the mold in the cryostat and
quickly frozen---just enough to make the muscle/frozen OCT strong enough
to survive removal from the mold. 3:  The tissue is removed from the
mold and placed on a freezing station in the cryostat.  An object
holder, kept at room temperature, is put into the cryostat on a freezing
station.  A blob of OCT is quickly put on the object holder and the
tissue in the frozen OCT/muscle block is stood up in the OCT---oriented
to stand up so that cross sections can be cut. The OCT will quickly
freeze holding the block in its cutting position. 4. The standing block
now needs to be bolstered with OCT for cutting.  OCT is added in
discrete steps and quickly frozen, building up by degrees a rectangle of
frozen OCT. The object holder with the block on it is then put in the
cutting position.  It is trimmed to make all edges of the rectangle
surrounding the tissue straight and clean and the corners square. The
block is positioned in the cryostat to make the long edge of the block
parallel with the knife edge. Sections are cut at 8 microns to nearly
fill the slide, and one section, 10 microns thick, completes the filling
of the slide.
Thanks, Mauricio.              


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