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<p class=MsoNormal><font size=3 color=navy face="Times New Roman"><span
style='font-size:12.0pt;color:navy'>What tissue are you after?<span
style='mso-spacerun:yes'> </span>For brain, I used a modified version of
the EM protocol published by <span class=SpellE>Cragg</span>, 1980.<span
style='mso-spacerun:yes'> </span>He found a way to avoid shrinkage due to
formalin fixation.<span style='mso-spacerun:yes'> </span><o:p></o:p></span></font></p>
<p class=MsoNormal><font size=3 color=navy face="Times New Roman"><span
style='font-size:12.0pt;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=3 color=navy face="Times New Roman"><span
style='font-size:12.0pt;color:navy'>When the fixative arrives, the sodium pump
proteins on the outside of the cell are the first to be fixed. <span
style='mso-spacerun:yes'> </span>Sodium rushes into the cell down the
concentration gradient, and water rushes in to maintain tonicity.<span
style='mso-spacerun:yes'> </span>The cell swells 30%, and fixation occurs
with <span class=SpellE>crosslinking</span> to proteins in neighboring cells.<span
style='mso-spacerun:yes'> </span>Later, when equilibrium is restored and the
cells shrink back, they drag their neighbors back with them.<span
style='mso-spacerun:yes'> </span>The result is 20% gross shrinkage of the
brain, and loss of <span class=SpellE>extracellular</span> space. <span
style='mso-spacerun:yes'> </span><o:p></o:p></span></font></p>
<p class=MsoNormal><font size=3 color=navy face="Times New Roman"><span
style='font-size:12.0pt;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><span class=SpellE><font size=3 color=navy
face="Times New Roman"><span style='font-size:12.0pt;color:navy'>Cragg</span></font></span><font
color=navy><span style='color:navy'> reasoned that if you could replace the <span
class=SpellE>extracellular</span> fluid with isotonic fluid without sodium, the
shrinkage would not occur at all.<span style='mso-spacerun:yes'>
</span>Isotonic sucrose (close to 5%) fills the bill, but will not replace <span
class=SpellE>extracellular</span> fluid because of the blood brain
barrier.<span style='mso-spacerun:yes'> </span>This can be overcome if
the sucrose is pumped through at 300 mm Hg to overcome the blood brain barrier
without breaking blood vessels. <span style='mso-spacerun:yes'> </span><o:p></o:p></span></font></p>
<p class=MsoNormal><font size=3 color=navy face="Times New Roman"><span
style='font-size:12.0pt;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=3 color=navy face="Times New Roman"><span
style='font-size:12.0pt;color:navy'>We offer a suitable apparatus to implement
this plan, see the link below, and a protocol.<span
style='mso-spacerun:yes'> </span>It has the salutary effect of completely
and thoroughly washing out the red blood cells.<span
style='mso-spacerun:yes'> </span>You use sucrose for the <span
class=SpellE>prewash</span> instead of saline, very briefly because of the high
pressure and flow rate, <span class=GramE>then</span> get the fixative to the cells
more rapidly than traditional methods.<span style='mso-spacerun:yes'>
</span><o:p></o:p></span></font></p>
<p class=MsoNormal><font size=3 color=navy face="Times New Roman"><span
style='font-size:12.0pt;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=3 color=navy face="Times New Roman"><span
style='font-size:12.0pt;color:navy'>Gravity flow is too weak, 150 mm Hg
translates to 6.7 ft of water.<span style='mso-spacerun:yes'> </span>In
your lab space, can the water reservoir be 6.7 ft above the animal on the sink,
without raising the ceiling?<span style='mso-spacerun:yes'> </span>Or to
implement the <span class=SpellE>Cragg</span> perfusion, you would need 13.4
ft. from animal to sucrose. <span
style='mso-spacerun:yes'> </span>Peristaltic pumps control flow
rate, which does not correct for vascular resistance of the animal, and the correct
flow rate is not obvious.<span style='mso-spacerun:yes'> </span>Try the
Perfusion One:<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=3 color=navy face="Times New Roman"><span
style='font-size:12.0pt;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=3 color=navy face="Times New Roman"><span
style='font-size:12.0pt;color:navy'><a
href="http://www.myneurolab.com/myneurolab/mnl_products_detail.asp?idproduct=471001&catdesc=Histology+Equipment&CatThreeID=674&CatOneID=4&subcatdesc=Sacrifice+Equipment&idsubcategory=21">http://www.myneurolab.com/myneurolab/mnl_products_detail.asp?idproduct=471001&catdesc=Histology+Equipment&CatThreeID=674&CatOneID=4&subcatdesc=Sacrifice+Equipment&idsubcategory=21</a><o:p></o:p></span></font></p>
<p class=MsoNormal><font size=3 color=navy face="Times New Roman"><span
style='font-size:12.0pt;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=3 color=navy face="Times New Roman"><span
style='font-size:12.0pt;color:navy'><o:p> </o:p></span></font></p>
<div>
<p><font size=3 color=navy face="Comic Sans MS"><span style='font-size:12.0pt;
font-family:"Comic Sans MS";color:navy;mso-no-proof:yes'>Charles W.
Scouten, Ph.D.</span></font><font color=navy><span style='color:navy;
mso-no-proof:yes'> <br>
</span></font><b><font color="#339966"><span style='color:#339966;font-weight:
bold;mso-no-proof:yes'>myNeuroLab.com</span></font></b><font color=navy><span
style='color:navy;mso-no-proof:yes'> <br>
</span></font><font size=2 color=navy face="Bookman Old Style"><span
style='font-size:10.0pt;font-family:"Bookman Old Style";color:navy;mso-no-proof:
yes'>5918 Evergreen Blvd.</span></font><font color=navy><span style='color:
navy;mso-no-proof:yes'> <br>
</span></font><font size=2 color=navy face="Bookman Old Style"><span
style='font-size:10.0pt;font-family:"Bookman Old Style";color:navy;mso-no-proof:
yes'>St. Louis, MO 63134</span></font><font color=navy><span style='color:navy;
mso-no-proof:yes'> <br>
</span></font><font size=2 color=navy face="Bookman Old Style"><span
style='font-size:10.0pt;font-family:"Bookman Old Style";color:navy;mso-no-proof:
yes'>Ph: 314 522 0300 </span></font><font color=navy><span
style='color:navy;mso-no-proof:yes'><br>
</span></font><font size=2 color=navy face="Bookman Old Style"><span
style='font-size:10.0pt;font-family:"Bookman Old Style";color:navy;mso-no-proof:
yes'>FAX 314 522 0377</span></font><font color=navy><span
style='color:navy;mso-no-proof:yes'> <br>
<a href="mailto:cwscouten@myneruolab.com">cwscouten@myneurolab.com</a> <br>
www.myneurolab.com <o:p></o:p></span></font></p>
</div>
<p class=MsoNormal><font size=3 color=navy face="Times New Roman"><span
style='font-size:12.0pt;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Tahoma><span
style='font-size:10.0pt;font-family:Tahoma'>-----Original Message-----<br>
<b><span style='font-weight:bold'>From:</span></b> Noel D. Clark
[mailto:Noel.Clark@ORTHO.UAB.EDU] <br>
<b><span style='font-weight:bold'>Sent:</span></b> Wednesday, October 01, 2003
7:45 AM<br>
<b><span style='font-weight:bold'>To:</span></b> Histonet@lists.utsouthwestern.edu<br>
<b><span style='font-weight:bold'>Subject:</span></b> [Histonet] Perfusion
techniques in mice/rats</span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'><o:p> </o:p></span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Was hoping to get input on the
various perfusion techniques used by other research labs for rodents. As
always, any help is greatly appreciated. See you in Louisville!</span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'> </span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Thanks in advance,</span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Noel</span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'> </span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'> </span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'> </span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'> </span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Noel Clark, M.A., HTL (ASCP)</span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>1919 7th Ave South</span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Orthopaedic Research Laboratory</span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Center for Metabolic Bone Disease</span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>University of Alabama at Birmingham</span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Birmingham, Alabama 35294</span></font><o:p></o:p></p>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'> <o:p></o:p></span></font></p>
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