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<DIV>Thanks for information on perfusion without a pump.</DIV>
<DIV> </DIV>
<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>
<DIV> </DIV>
<BLOCKQUOTE dir=ltr style="MARGIN-RIGHT: 0px">
<DIV><FONT size=2>-----Original Message----- <BR><B>From:</B> Pamela Marcum
[mailto:pmarcum@polysciences.com] <BR><B>Sent:</B> Wed 10/1/2003 11:07 AM
<BR><B>To:</B> Davis, Gareth; Histonet <BR><B>Cc:</B> <BR><B>Subject:</B> RE:
[Histonet] Perfusion techniques in mice/rats<BR><BR></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff><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 face=Arial color=#0000ff 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="PADDING-LEFT: 5px; MARGIN-LEFT: 5px; BORDER-LEFT: #0000ff 2px solid; MARGIN-RIGHT: 0px"></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></STRONG> </P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt"><B>Preparation for
Perfusion:</B></P>
<P class=MsoNormal style="MARGIN: 0in 0in 0pt"><B></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> </SPAN><SPAN>
</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>
</SPAN>Add 38g of sodium borate, paraformaldehyde will not dissolve until it
is added.<SPAN> </SPAN>Cool off to 4<SUP>o</SUP>C.<SPAN>
</SPAN>Adjust pH to 9.5 with <B>glacial acetic acid.</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</B></P>
<OL style="MARGIN-TOP: 0in" type=1>
<LI class=MsoNormal style="MARGIN: 0in 0in 0pt">Anesthetize animal,
open thoracic cavity, expose heart and visualize ascending
aorta.<SPAN> </SPAN>Insert 18-gauge needle (on vacutainer collection
tubing) through<U> left ventricle</U> into ascending aorta- if possible,
hard to tell sometimes.<SPAN> </SPAN>Clamp cannula into place with
would clip or small clamp and <U>then snip right
atrium</U>.<SPAN> (<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"><SPAN></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> </SPAN>
<LI class=MsoNormal style="MARGIN: 0in 0in 0pt">Change to cold 4%
PFA and slowly perfuse animal.<SPAN> </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">After perfusion is
complete, decapitate the animal and quickly remove the brain using a
caudal approach.<SPAN> </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 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>
<DIV> </DIV></BLOCKQUOTE></BLOCKQUOTE></BODY></HTML>