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<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>1.<span style='mso-spacerun:yes'> </span>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.<span
style='mso-spacerun:yes'> </span>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.<span style='mso-spacerun:yes'> </span>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.<span
style='mso-spacerun:yes'> </span><span class=GramE>As much of each segment of
the tissue should be represented as well as possible in the sections.</span><o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>2.<span style='mso-spacerun:yes'> </span>OCT is the
mounting medium of choice.<span style='mso-spacerun:yes'> </span>It is compatible
with all of the <span class=SpellE>histochemistry</span>. Only a thin coating
of OCT is put in the bottom of the mold to hold the tissue in the mold during
freezing.<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>3.<span style='mso-spacerun:yes'> </span>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<span
class=GramE>,<span style='mso-spacerun:yes'> </span>and</span> the other a
rectangle of about an inch or so by half an inch <o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>4.<span style='mso-spacerun:yes'> </span>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.<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>5.<span style='mso-spacerun:yes'> </span>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
<span class=SpellE>isopentane</span> and lowered into the liquid nitrogen. The
cup is best held with long forceps.<span style='mso-spacerun:yes'> </span>When
the fizzing stops for the second time---there is usually a secondary roiling of
the <span class=SpellE>isopentane</span>----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.<span style='mso-spacerun:yes'> </span>This is done until you have. <span
class=GramE>a</span> 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 <span class=SpellE>isopentane</span>,
and put in the liquid nitrogen for 10- to 20 slow dunks.<span
style='mso-spacerun:yes'> </span>Then the mold is put directly into the
cryostat.<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>6.<span style='mso-spacerun:yes'> </span>The tissues are
removed from the mold inside the cryostat.<span style='mso-spacerun:yes'>
</span>The frozen OCT with the muscle in it is placed on a specimen holder for
your cryostat.<span style='mso-spacerun:yes'> </span>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.<span style='mso-spacerun:yes'> </span>Go watch the
residents sometimes, cutting <span class=SpellE>frozens</span> with the bare
tissue turned any which way, splitting, turning, getting cussed.<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>7.<span style='mso-spacerun:yes'> </span>Sections are put
onto 2 by 3 inch glass slides.<span style='mso-spacerun:yes'> </span>Using
cover glass is a volunteering for misery. I can’t imagine how that got
started.<span style='mso-spacerun:yes'> </span>You can handle and store the
glass slides easier and 178.56 times safer than when on cover glass.<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>8.<span style='mso-spacerun:yes'> </span>Reagents are mixed
to FILL the <span class=SpellE>coplin</span> jars, completely covering the
tissues on the slides.<span style='mso-spacerun:yes'> </span>Stingy volumes <span
class=GramE>of<span style='mso-spacerun:yes'> </span><span class=SpellE>histochemical</span></span>
preparations is inherited thru the genes from Scrooge!!<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>9.<span style='mso-spacerun:yes'> </span>Sigma sells a
buffer called 221 that you will see used in the ATP-<span class=SpellE>ase</span>
and the other preparations in the packet.<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>.<span style='mso-spacerun:yes'> </span>It does not require
a controlled substance license to purchase and it beats the socks off the
sodium pentobarbital in all functions.<span style='mso-spacerun:yes'>
</span><span style='mso-tab-count:1'> </span><span
style='mso-spacerun:yes'> </span><span
style='mso-spacerun:yes'> </span><o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>All of this is better covered in the video.<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Now then, what’s going to happen?<span
style='mso-spacerun:yes'> </span>What will win out there in your labs----habit
or function??<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p> </o:p></span></font></p>
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