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<DIV><SPAN class=533341816-28022001><FONT face=Arial color=#0000ff size=2>In
order to permeabilize the nuclear membrane of frozen tissue (or cytology
specimens) when staining for ER & PR, one needs to use either Zamboni's
fixative (a picric acid fixative) or the 3 step fixation protocol I
mentioned:</FONT></SPAN></DIV>
<DIV><SPAN class=533341816-28022001><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=533341816-28022001><FONT face=Arial color=#0000ff size=2>Place
freshly cut frozen section slides in 10% NBF for 10-15". Transfer to
buffer (Tris, PBS, etc.) for a minimum of 5". Transfer slides to cold
methanol for 3-5"; then to cold acetone for 1-3". Place slides in buffer
for a maximum of 2 hours or store at -20 degrees C in specimen storage medium if
not staining within 2 hours.</FONT></SPAN></DIV>
<DIV><SPAN class=533341816-28022001><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=533341816-28022001><FONT face=Arial color=#0000ff
size=2>Specimen Storage Medium:</FONT></SPAN></DIV>
<DIV><SPAN class=533341816-28022001><FONT face=Arial color=#0000ff
size=2>Dissolve 42.8g sucrose and 0.70g magnesium chloride in PBS to a volume of
250ml. Add 250 ml glycerol and mix well.</FONT></SPAN></DIV>
<DIV><SPAN class=533341816-28022001><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=533341816-28022001><FONT face=Arial color=#0000ff size=2>We
recently stained frozen section slides that had been fixed with the 3 step
method and then stored in this medium at -20 degrees C for <U>several years</U>;
the staining was as intense as ever!</FONT></SPAN></DIV>
<DIV><SPAN class=533341816-28022001><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=533341816-28022001><FONT face=Arial color=#0000ff
size=2>Ref.: <U>Abbott ER-ECA Monoclonal Immunocytochemical Assay for the
Detection of Human Estrogen Receptor</U> product insert, Abbott Laboratories,
Diagnostics Division</FONT></SPAN></DIV>
<DIV><FONT face=Arial color=#0000ff size=2></FONT> </DIV>
<P><FONT face=Arial size=2>Linda A. Sebree, HT(ASCP)</FONT> <BR><FONT face=Arial
size=2>University of Wisconsin Hospital & Clinics</FONT> <BR><FONT
face=Arial size=2>IHC/ISH Laboratory</FONT> <BR><FONT face=Arial
size=2>A4/204-2472</FONT> <BR><FONT face=Arial size=2>600 Highland Ave.</FONT>
<BR><FONT face=Arial size=2>Madison, WI 53792-2472</FONT> <BR><FONT face=Arial
size=2>(608)265-6596</FONT> <BR><FONT face=Arial size=2>FAX:
(608)262-7174</FONT> </P>
<BLOCKQUOTE>
<DIV class=OutlookMessageHeader dir=ltr align=left><FONT face=Tahoma
size=2>-----Original Message-----<BR><B>From:</B> Elizaha@aol.com
[mailto:Elizaha@aol.com]<BR><B>Sent:</B> Tuesday, February 27, 2001 8:33
PM<BR><B>To:</B> la.sebree@hosp.wisc.edu<BR><B>Subject:</B> Re: HIER on frozen
tissue sections<BR><BR></FONT></DIV><FONT face=arial,helvetica><FONT
size=2>Hi, <BR> What is the three step method all
about?</FONT> </FONT></BLOCKQUOTE></BODY></HTML>