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<DIV><FONT color=#0000ff face=Arial><SPAN class=46445415-01102003>If you do not
have a pump available you can use an old fashion method. A glass
bottle and stopper with two holes - one for the tubing and 18 gauge
needle, sharp edge filed smooth (or size required) to be attached
through and one for air to be admitted that is longer and is above the fluid
level. Or a refillable IV bag from the pharmacy with tubing.
The</SPAN></FONT><FONT face="Times New Roman" size=2><SPAN
class=46445415-01102003><FONT color=#0000ff face=Arial size=3> bag or
bottle is hung at least 2 1/2 feet to 3 feet above the level of the animal to be
perfused so make sure the tubing is long enough. The clamp on the tubing
for fluid should be adjustable and set for a steady and slow drip to avoid
vascular damage by rapid rate of flow. The methods for opening the
chest and exposure of the heart are clear in all of the suggestions given.
The descending aorta can be clamped to avoid perfusing the lower part of the
body. (One thing about using a bottle - Remember to place rubber bands
around the bottle over the stopper as they can come out and fixative goes
everywhere.) Pam Marcum</FONT></SPAN></FONT></DIV>
<DIV><FONT face="Times New Roman" size=2><SPAN
class=46445415-01102003></SPAN></FONT> </DIV>
<DIV><FONT face="Times New Roman" size=2><SPAN
class=46445415-01102003></SPAN></FONT> </DIV>
<DIV><FONT face="Times New Roman" size=2><SPAN
class=46445415-01102003> </SPAN>-----Original Message-----<BR><B>From:</B>
histonet-admin@lists.utsouthwestern.edu
[mailto:histonet-admin@lists.utsouthwestern.edu]<B>On Behalf Of</B> Davis,
Gareth<BR><B>Sent:</B> Wednesday, October 01, 2003 10:32 AM<BR><B>To:</B>
Histonet<BR><B>Subject:</B> Re: [Histonet] Perfusion techniques in
mice/rats<BR><BR></DIV>
<BLOCKQUOTE
style="BORDER-LEFT: #0000ff 2px solid; MARGIN-LEFT: 5px; MARGIN-RIGHT: 0px; PADDING-LEFT: 5px"></FONT>
<DIV>
<P class=MsoTitle style="MARGIN: 0in 0in 0pt"><STRONG><U><FONT
size=5>Perfusion Protocol</FONT></U></STRONG></P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt"><STRONG> <?xml:namespace
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 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 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 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></DIV>
<DIV><FONT color=#800080 face=System size=2>Ms. Gareth B. Davis</FONT></DIV>
<DIV><FONT color=#800080 face=System size=2><STRONG>Research Assistant
II</STRONG></FONT></DIV>
<DIV><FONT color=#800080 face=System size=2>Neuro-magnetics Division of
</FONT></DIV>
<DIV><FONT color=#800080 face=System size=2>the Department of
Neurology</FONT></DIV>
<DIV><FONT color=#800080 face=System size=2>Vanderbilt University Medical
Center</FONT></DIV>
<DIV><FONT color=#800080 face=System size=2>615-936-3318</FONT></DIV>
<DIV><FONT color=#800080 face=System size=2></FONT> </DIV>
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