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<DIV><FONT face=Tahoma><FONT size=2>Margarete<SPAN
class=770171123-30092003>,</SPAN></FONT></FONT></DIV>
<DIV><FONT face=Tahoma><FONT size=2><SPAN
class=770171123-30092003></SPAN></FONT></FONT> </DIV>
<DIV><FONT face=Tahoma><FONT size=2><SPAN class=770171123-30092003>ISH on frozen
sections is possible. I have had success with the following to demonstrate mRNA.
The nucleolus stains up beautifully:</SPAN></FONT></FONT></DIV><SPAN
class=770171123-30092003><B>
<P><FONT face=Tahoma size=2>SOLUTIONS:</FONT></P></B>
<P><FONT face=Tahoma size=2>1. Acetic Alcohol</FONT></P>
<P><FONT face=Tahoma size=2>Absolute Alcohol 15ml</FONT></P>
<P><FONT face=Tahoma size=2>Acetic Acid 5ml</FONT></P>
<P><FONT face=Tahoma size=2>2. Absolute Alcohol</FONT></P>
<P><FONT face=Tahoma size=2>3. Cold Acetone</FONT></P>
<P><B><FONT face=Tahoma size=2>METHOD:</FONT></P></B>
<OL>
<LI><FONT face=Tahoma size=2>Air dry frozen sections (6-8um) minimum 1
hour.</FONT></LI>
<LI><FONT face=Tahoma size=2>Fix in Acetic Alcohol 15 minutes.</FONT></LI>
<LI><FONT face=Tahoma size=2>Wash in absolute alcohol.</FONT></LI>
<LI><FONT face=Tahoma size=2>Place in cold acetone, allow to warm to room
temperature.</FONT></LI>
<LI><FONT face=Tahoma size=2>Remove slides and air dry.</FONT></LI>
<LI><FONT face=Tahoma size=2>Circle sections with PAP pen, add probe and
continue with ISH.</FONT></LI></OL>
<DIV><SPAN class=770171123-30092003><FONT face=Tahoma size=2>NB do not treat
with enzyme or acid.</FONT></SPAN></DIV>
<DIV><SPAN class=770171123-30092003><FONT face=Tahoma size=2>I would suggest
sealing the coverslip with nail polish (after adding th probe) and heating on a
hot plate to denature the DNA if searching for DNA
sequences.</FONT></SPAN></DIV>
<DIV><SPAN class=770171123-30092003><FONT face=Tahoma
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=770171123-30092003>
<P><FONT face=Arial size=2>Tony Henwood JP, B</FONT><FONT face=Arial size=2>A</FONT><FONT face=Arial size=2>ppSc, GradDipSysAnalys, CT(ASC)</FONT>
<BR><FONT face=Arial size=2>Laboratory Manager</FONT> <BR><FONT face=Arial
size=2>The Children's Hospital at Westmead,</FONT> <BR><FONT face=Arial
size=2>Locked Bag 4001, Westmead, 2145, AUSTRALIA.</FONT> <BR><FONT face=Arial
size=2>Tel:</FONT> <FONT face=Arial size=2>61</FONT><FONT face=Arial size=2>2
9845 3306</FONT> <BR><FONT face=Arial size=2>Fax:</FONT> <FONT face=Arial
size=2>61</FONT><FONT face=Arial size=2>2 9845 3318</FONT> </P>
<P><FONT face=Arial size=2><A target=_blank
href="http://www.histosearch.com/homepages/TonyHenwood/default.html">http://www.histosearch.com/homepages/TonyHenwood/default.html</A></FONT>
<BR><FONT face=Arial size=2><A target=_blank
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<DIV class=OutlookMessageHeader dir=ltr align=left><FONT face=Tahoma
size=2>-----Original Message-----<BR><B>From:</B> Margarete Vermehren [mailto:m.vermehren@anat.vetmed.uni-muenchen.de]<BR><B>Sent:</B> Tuesday, 30
September 2003 23:57<BR><B>To:</B>
histonet@lists.utsouthwestern.edu<BR><B>Subject:</B> [Histonet] ISH on fresh
frozen tissue<BR><BR></FONT></DIV>
<DIV class=Section1>
<P class=MsoNormal><SPAN class=EmailFormatvorlage16><FONT face=Tahoma
size=2><SPAN lang=EN-GB
style="FONT-SIZE: 11pt; FONT-FAMILY: Tahoma; mso-bidi-font-size: 12.0pt; mso-bidi-font-family: 'Times New Roman'">I
am trying to do ISH on fresh frozen mouse testis. The morphology was o.k. but
my protocol doesn’t work. This material should not need pretreatment, I
suppose, although I must be wrong somewhere – it doesn’t work that way.
Pretreatment with protease E is very delicate. Has anybody got experience he/she could share with me?<o:p></o:p></SPAN></FONT></SPAN></P>
<P class=MsoNormal><SPAN class=EmailFormatvorlage16><FONT face=Tahoma
size=2><SPAN lang=EN-GB
style="FONT-SIZE: 11pt; FONT-FAMILY: Tahoma; mso-bidi-font-size: 12.0pt; mso-bidi-font-family: 'Times New Roman'">Thank
you very much!!<o:p></o:p></SPAN></FONT></SPAN></P>
<P class=MsoNormal><SPAN class=EmailFormatvorlage16><FONT face=Tahoma
size=2><SPAN lang=EN-GB
style="FONT-SIZE: 11pt; FONT-FAMILY: Tahoma; mso-bidi-font-size: 12.0pt; mso-bidi-font-family: 'Times New Roman'">Margaret</SPAN></FONT></SPAN><SPAN
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