[Histonet] Suggested corrections for list of errors

[George Cole]

1.  ALL of the tissues sent will be frozen and sectioned. There is no
way to look at 100% of the tissues---but we can represent every portion
of the tissues.  With normally sized biopsies, pieces to be cut may be
just slightly narrower than the cover glass or a group of several narrow
pieces just narrower than the cover glass can be frozen side by side.
Pieces are best processed if they are no more than half a cm thick and
long.  Huge biopsies a cubic inch and larger occasionally arrive. Pieces
a cm long and almost a cm thick have been frozen all right, with
occasional minor ice crystals in the center.  As much of each segment of
the tissue should be represented as well as possible in the sections.
2.  OCT is the mounting medium of choice.  It is compatible with all of
the histochemistry. Only a thin coating of OCT is put in the bottom of
the mold to hold the tissue in the mold during freezing.
3.  Those thin walled plastic molds work just fine---they have a name,
but I never knew it--- They come in at least two different sizes. About
a ¾ inch square cavity,  and the other a rectangle of about an inch or
so by half an inch 
4.  A thin coating of OCT is put on the bottom of the mold to act as
glue, so the tissue won’t pop out during freezing.
5.  A thin walled aluminum cup----2.75 to 3 inches diameter at the brim
and slightly narrower at the bottom (mine was meant to be a gravy mixer)
is filled with a cup or more of isopentane and lowered into the liquid
nitrogen. The cup is best held with long forceps.  When the fizzing
stops for the second time---there is usually a secondary roiling of the
isopentane----a SMALL bit of liquid nitrogen is admitted into the cup (
No need my telling you a small bit---use more---you won’t want to do it
twice---it boils all over the place! ) and stirred. I used a butter
knife with thick wrapping of paper towels around the handle---it gets
COLD.  This is done until you have. a thick slurry. The muscle in the
mold is quickly put into the slurry and dunked to a slow count of 10 to
20. The mold is lifted out of the slurry, shaken twice quickly, to shake
off the clinging isopentane, and put in the liquid nitrogen for 10- to
20 slow dunks.  Then the mold is put directly into the cryostat.
6.  The tissues are removed from the mold inside the cryostat.  The
frozen OCT with the muscle in it is placed on a specimen holder for your
cryostat.  It is then bolstered about with OCT ---the bolstering results
in a neat rectangle wider across than up and down. All of the edges are
trimmed to be straight and clean---a straight edged, square cornered
rectangle which is cut with the broad bottom to the knife. You will see
that with edges straight and neat, you will have happier time cutting
sections.  Go watch the residents sometimes, cutting frozens with the
bare tissue turned any which way, splitting, turning, getting cussed.
7.  Sections are put onto 2 by 3 inch glass slides.  Using cover glass
is a volunteering for misery. I can’t imagine how that got started.  You
can handle and store the glass slides easier and 178.56 times safer than
when on cover glass.
8.  Reagents are mixed to FILL the coplin jars, completely covering the
tissues on the slides.  Stingy volumes of  histochemical preparations is
inherited thru the genes from Scrooge!!
9.  Sigma sells a buffer called 221 that you will see used in the
ATP-ase and the other preparations in the packet.
.  It does not require a controlled substance license to purchase and it
beats the socks off the sodium pentobarbital in all functions.

 
All of this is better covered in the video.
Now then, what’s going to happen?  What will win out there in your
labs----habit or function??
 
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