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<DIV><SPAN class=414255822-19112003><FONT face=Arial color=#0000ff size=2>Sorry,
I neglected to mention - </FONT></SPAN></DIV>
<DIV><SPAN class=414255822-19112003><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=414255822-19112003><FONT face=Arial color=#0000ff size=2>I have
also tried </FONT></SPAN></DIV>
<DIV><SPAN class=414255822-19112003><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=414255822-19112003><FONT face=Arial color=#0000ff
size=2>Sections were cut in cryostat and stored as unfixed slides overnight in
-80. (no dessication though)</FONT></SPAN></DIV>
<DIV><SPAN class=414255822-19112003><FONT face=Arial color=#0000ff
size=2>Unfixed frozen slides (stored at -80) placed at -20 for 5
minutes</FONT></SPAN></DIV>
<DIV><SPAN class=414255822-19112003><FONT face=Arial color=#0000ff size=2>Placed
in -20 acetone for 5 minutes.</FONT></SPAN></DIV>
<DIV><SPAN class=414255822-19112003><FONT face=Arial color=#0000ff size=2>Air
dried at room temperature for 10 minutes</FONT></SPAN></DIV><SPAN
class=414255822-19112003>
<DIV><FONT face=Arial color=#0000ff size=2></FONT><BR><FONT face=Arial
color=#0000ff size=2><SPAN class=414255822-19112003>As per standard
protocol.</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2></FONT></SPAN><SPAN
class=414255822-19112003><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<BLOCKQUOTE style="MARGIN-RIGHT: 0px">
<DIV></DIV>
<DIV class=OutlookMessageHeader lang=en-us dir=ltr align=left><FONT
face=Tahoma size=2>-----Original Message-----<BR><B>From:</B> Tan, MinHan
<BR><B>Sent:</B> Wednesday, November 19, 2003 5:33 PM<BR><B>To:</B>
histonet@lists.utsouthwestern.edu<BR><B>Subject:</B> [Histonet] Frozen section
fixing problems - but paraffin works!<BR><BR></FONT></DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2>Hi,</FONT></SPAN></DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial size=2>I am working with
a new monoclonal antibody in parathyroid tissue, and I use the ABC-DAB
reageant system for immunohistochemistry.</FONT></SPAN></DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial size=2>I am new to frozen
sections immunostaining - and I am encountering problems with frozen sections
- staining is very weak or absent, in comparison to paraffin embedded tissue,
which I have no problems with. </FONT></SPAN></DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=664142622-19112003></SPAN><SPAN
class=664142622-19112003><FONT face=Arial size=2>My protocol for frozen
sections: </FONT></SPAN></DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial size=2>Unfixed frozen
slides with 5 micron sections thawed at room temperature for 5
minutes</FONT></SPAN></DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial size=2>Placed in 70%
ethanol x 5 minutes.</FONT></SPAN></DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial size=2>Washed in PBS x 5
minutes.</FONT></SPAN></DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2>...</FONT></SPAN></DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial size=2>followed with
standard protocol: hydrogen peroxide 0.3% x 30 min, donkey serum 5% x 30
min, primary antibody (mouse) 4 deg overnight, secondary (goat anti-mouse),
ABC, DAB.</FONT></SPAN></DIV>
<DIV><SPAN class=664142622-19112003></SPAN><SPAN
class=664142622-19112003></SPAN> </DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial size=2>Does anyone have
any advice?</FONT></SPAN></DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial size=2>Thank
you!</FONT></SPAN></DIV><SPAN class=664142622-19112003>
<DIV><FONT face=Arial size=2></FONT><FONT face=Arial size=2></FONT><FONT
face=Arial size=2></FONT><FONT face=Arial size=2></FONT><FONT face=Arial
size=2></FONT><FONT face=Arial size=2></FONT><FONT face=Arial
size=2></FONT><FONT face=Arial size=2></FONT><BR><FONT face=Arial
size=2>Regards,</FONT></DIV>
<DIV><FONT face=Arial><FONT size=2><SPAN class=664142622-19112003>Min-Han
Tan</SPAN> </FONT></FONT></DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2></FONT></SPAN> </DIV></SPAN>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=664142622-19112003><FONT face=Arial
size=2></FONT></SPAN> </DIV><FONT face=Arial>
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