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<DIV><SPAN class=485173105-11122003><FONT face=Arial size=2>Hi there
Histonetters,</FONT></SPAN></DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial size=2>I would like to seek
your advice about a problem I am having with frozen
sections.</FONT></SPAN></DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial size=2>I am working on some
frozen tissue dating back to about 1998, and performing IHC on parathyroid
tissue with p27 antibody. I use the DAB-ABC method.</FONT></SPAN></DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial size=2>I have noted that
certain samples (and particular samples only) exhibit loss of nuclei in the
central region (an amorphous blob of tissue extending to near the margins, about
70-80% of the specimen) (no staining with hematoxylin either). Any nuclei that
stain with my IHC in that area are very faint, and there is no
hematoxylin counterstained nuclei in the entire region. Nuclear staining
seems to be fine near the margins - of course it can be argued that these are
artifacts. Where there are folds of tissue, both nuclear and
background staining is prominent. Other samples of the same type of tissue
seem to stain OK throughout the tissue. No problems with paraffin-embedded
tissue at all, the nucleus stains fine in every sample I have tried.
</FONT></SPAN></DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial size=2>It is unlikely to be
due to underfixation, since I immerse my 5 micron unfixed slide in
neutral buffered formalin for 30 min prior to IHC. I see it regardless of
whether I perform HIER on my frozen sections or not. (yes, I do perform HIER on
my frozen sections)</FONT></SPAN></DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial size=2>I wonder if the
following explanations are plausible:<BR>(a) tissue has degraded - but it would
not make sense for degradation to take place from inside
out.</FONT></SPAN></DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial size=2>(b) sections may be
too thin for some reason? (mine are 5 micron) - but this might explain why
only the borders and folds stain.</FONT></SPAN></DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial size=2>Thank
you!<BR><BR>Min-Han Tan</FONT></SPAN></DIV>
<DIV><SPAN class=485173105-11122003><FONT face=Arial size=2>Van Andel Research
Institute</FONT></SPAN></DIV>
<FONT face=Arial><HR><STRONG></FONT>
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