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<P class=MsoTitle style="MARGIN: 0in 0in 0pt"><STRONG><U><FONT size=5>Perfusion
Protocol</FONT></U></STRONG></P>
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prefix = o ns = "urn:schemas-microsoft-com:office:office"
/><o:p></o:p></STRONG></P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt"><B>Preparation for
Perfusion:<o:p></o:p></B></P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt"><B><o:p></o:p></B> </P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt"><B> </B><B>Fixative =4%
PFA </B>Prepare fresh on day of use or the night before and keep at
4<SUP>o</SUP>C.</P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt 0.5in; TEXT-INDENT: 0.5in">4%
Paraformaldehyde</P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt"><SPAN
style="mso-tab-count: 1"> </SPAN><SPAN
style="mso-tab-count: 1">
</SPAN>3.8% Sodium Borate</P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt">Add 40g of paraformaldehyde to
800ml of water and heat to 60<SUP>o</SUP>C while stirring.<SPAN
style="mso-spacerun: yes"> </SPAN>Add 38g of sodium borate,
paraformaldehyde will not dissolve until it is added.<SPAN
style="mso-spacerun: yes"> </SPAN>Cool off to 4<SUP>o</SUP>C.<SPAN
style="mso-spacerun: yes"> </SPAN>Adjust pH to 9.5 with <B>glacial acetic
acid.<o:p></o:p></B></P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt"><B> 4% PFA with 10%
sucrose=</B></P>
<P class=MsoNormal
style="MARGIN: 0in 0in 0pt"><STRONG>
</STRONG>In a 100ml cylinder add 10g of Sucrose, than add 4% PFA to 100ml
</P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt"> </P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt"> </P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt"><B>Perfusion<o:p></o:p></B></P>
<OL style="MARGIN-TOP: 0in" type=1>
<LI class=MsoNormal
style="MARGIN: 0in 0in 0pt; mso-list: l0 level1 lfo1; tab-stops: list .5in">Anesthetize animal,
open thoracic cavity, expose heart and visualize ascending aorta.<SPAN
style="mso-spacerun: yes"> </SPAN>Insert 18-gauge needle (on vacutainer
collection tubing) through<U> left ventricle</U> into ascending aorta- if
possible, hard to tell sometimes.<SPAN style="mso-spacerun: yes">
</SPAN>Clamp cannula into place with would clip or small clamp and
<U>then snip right atrium</U>.<SPAN
style="mso-spacerun: yes"> (<STRONG>I find it's easier - on mice- to hold
needle in place, because clamp gets in the way</STRONG>). </SPAN></LI>
<LI class=MsoNormal
style="MARGIN: 0in 0in 0pt; mso-list: l0 level1 lfo1; tab-stops: list .5in"><SPAN
style="mso-spacerun: yes"></SPAN>Slowly perfuse animal with about 40-50ml room
temperature saline <STRONG>(it's recommended to use a pump here, but I
use 50 ml syringe, and perfuse slowly)</STRONG> until fluid
is clear<STRONG>.</STRONG><SPAN
style="mso-spacerun: yes"> </SPAN></LI>
<LI class=MsoNormal
style="MARGIN: 0in 0in 0pt; mso-list: l0 level1 lfo1; tab-stops: list .5in">Change
to cold 4% PFA and slowly perfuse animal.<SPAN
style="mso-spacerun: yes"> </SPAN>When the animal’s forelimbs are fully
extended reduce perfusion speed, use about 50ml of fixative (for a 30g
mouse).</LI>
<LI class=MsoNormal
style="MARGIN: 0in 0in 0pt; mso-list: l0 level1 lfo1; tab-stops: list .5in">After
perfusion is complete, decapitate the animal and quickly remove the brain
using a caudal approach.<SPAN style="mso-spacerun: yes"> </SPAN>Place
brain in 4% PFA with 10% sucrose at 4<SUP>o</SUP>C for overnight. (The
4%PFA with sucrose will post-fix brain and cryoprotect tissue at the same
time.) <STRONG>Some recommend to post-fix in 4% PFA-only, overnight,
then cryoprotect with 30% sucrose for another 24 hours</STRONG>. </LI></OL>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt"><B> <o:p></o:p></B></P>
<DIV><FONT face=System color=#800080 size=2>Ms. Gareth B. Davis</FONT></DIV>
<DIV><FONT face=System color=#800080 size=2><STRONG>Research Assistant
II</STRONG></FONT></DIV>
<DIV><FONT face=System color=#800080 size=2>Neuro-magnetics Division of
</FONT></DIV>
<DIV><FONT face=System color=#800080 size=2>the Department of
Neurology</FONT></DIV>
<DIV><FONT face=System color=#800080 size=2>Vanderbilt University Medical
Center</FONT></DIV>
<DIV><FONT face=System color=#800080 size=2>615-936-3318</FONT></DIV>
<DIV><FONT face=System color=#800080 size=2></FONT> </DIV>
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