<!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0 Transitional//EN">
<HTML><HEAD><TITLE>Message</TITLE>
<META http-equiv=Content-Type content="text/html; charset=us-ascii">
<META content="MSHTML 6.00.2800.1276" name=GENERATOR></HEAD>
<BODY>
<DIV><SPAN class=239030117-02122003><FONT face=Arial color=#0000ff size=2>Dear
Russ,</FONT></SPAN></DIV>
<DIV><SPAN class=239030117-02122003> <FONT face=Arial
color=#0000ff size=2>Back in the '60's, I routinely counterstained the PAS with
H&E, the magenta of the Basic Fuchsin is not confused with the orange/red of
the Eosin. Also, since I always use alcoholic Eosin, that which can be
extracted from the tissue will dissolve quite nicely in subsequent
rinses in 95% EtOH. Eosin tends to be less soluble in 100% EtOH than in
95%. </FONT></SPAN></DIV>
<DIV><SPAN class=239030117-02122003> <FONT face=Arial
color=#0000ff size=2>Now, to the matter of converting an H&E to a
PAS.</FONT></SPAN></DIV>
<DIV><SPAN class=239030117-02122003> <FONT face=Arial
color=#0000ff size=2>My take on your request is that you would like to bleach an
H&E, and reprocess as PAS. My working hypothesis would be as
follows. Since the PAS preparations with which I am familiar are already
quite acidic, I would expect any H in the section to be
destabilized, to the extent possible, by the PA first and then the S
themselves. Alcohols and water should take care of that part of the E that
can be removed. </FONT></SPAN></DIV>
<DIV><SPAN class=239030117-02122003><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=239030117-02122003><FONT face=Arial color=#0000ff
size=2> In contrast to J. Kiernan, with whom I take exception
only with great trepidation, I would use no other than the following,
which I would describe as a relatively straight forward -
Protocol.</FONT></SPAN></DIV>
<DIV><SPAN class=239030117-02122003><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN
class=239030117-02122003> <FONT
face=Arial color=#0000ff size=2>1. Soak slides in Xylene
to remove coverglass and excess mountant. Five slides in 25-35 degrees 'C'
Xylene for four hours, tease the coverglasses away with #11 scalpel blade
and then leave overnight.</FONT></SPAN></DIV>
<DIV><SPAN
class=239030117-02122003> <FONT
face=Arial color=#0000ff size=2>2. Soak again to insure
complete removal of mountant. Thus, two more fresh Xylene rinses
for 8 and 16 hours respectively.</FONT> </SPAN></DIV>
<DIV><SPAN class=239030117-02122003>
<FONT face=Arial color=#0000ff size=2>3. Bring sections slowly
to water. </FONT></SPAN></DIV>
<DIV><SPAN class=239030117-02122003><FONT face=Arial color=#0000ff
size=2>
a. Excess times in ethanols will have no deleterious effect on
PAS positivity, so again rinse in several Coplin jars of 100%, then 95%, then
70% EtOH. Two to three hours should provide sufficient time in a total of
six alcohols (2x100, 2x95 & 2x70)</FONT></SPAN></DIV>
<DIV><SPAN
class=239030117-02122003> <FONT
face=Arial color=#0000ff size=2>b. Any touch of milkiness
in sections once they are in 70% is evidence of residual xylene. Go back
to 100% and re-hydrate.</FONT></SPAN></DIV>
<DIV><SPAN class=239030117-02122003>
<FONT face=Arial color=#0000ff size=2>4. Immerse in 1%
periodic acid for 10 min at RT.</FONT></SPAN></DIV>
<DIV><SPAN
class=239030117-02122003> <FONT
face=Arial><FONT color=#0000ff size=2>5. My PAS uses the
de'Tomasi version of the Schiff's ('S') reagent (found in Pearse, all
editions).</FONT></FONT> <FONT face=Arial color=#0000ff size=2>Immerse for
10 min at RT, though I have left for up to 20 min without any observable
negative effect.</FONT></SPAN></DIV>
<DIV><SPAN class=239030117-02122003>
<FONT face=Arial color=#0000ff size=2>6. Rinse at least 2X in
HOH (always d.i. or distilled). HOH should demonstrate little if any pink
color, though on standing some color will appear.</FONT></SPAN></DIV>
<DIV><SPAN class=239030117-02122003>
<FONT face=Arial color=#0000ff size=2>7. Rinse in 'gently'
running tap water (Lehigh or Delaware Valley's of Pennsylvania, if possible) for
5 min. [This step is ALL about modern alchemistery [Sic(k?)!], no matter
in what procedure it is used!] The section should 'pink' (magenta?) up the
section.</FONT></SPAN></DIV>
<DIV><SPAN class=239030117-02122003>
<FONT face=Arial color=#0000ff size=2>8. Quickly view with
cover glass (HOH mounted) and green filter (Wratten #58 is my favorite).
This step will help to certify the PAS and to determine advisability
of re-countering with H and/or E. Unless I were running parallel sections
as PAS controls, I would always counter with
H&E. </FONT></SPAN></DIV>
<DIV><SPAN class=239030117-02122003><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=239030117-02122003><FONT face=Arial color=#0000ff
size=2>NOTE: I counter with H&E after I process sections or air-dried
mesentery spreads in Gomori's Aldehyde Fuchsin for
elastin.</FONT></SPAN></DIV>
<DIV><SPAN class=239030117-02122003><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=239030117-02122003>
<FONT face=Arial color=#0000ff size=2>9. Mount coverglass as
required.</FONT></SPAN></DIV>
<DIV><SPAN class=239030117-02122003><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=239030117-02122003></SPAN><FONT face=Arial color=#0000ff
size=2><SPAN class=239030117-02122003>Hope this helps,</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=239030117-02122003></SPAN></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=239030117-02122003>Fred
Monson (Never known for brevity!)</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2></FONT> </DIV><!-- Converted from text/plain format -->
<P><FONT size=2> Frederick C. Monson, PhD<BR>Center for Advanced Scientific
Imaging<BR>Mail to Geology<BR>West Chester University of
Pennsylvania<BR>Schmucker II Science Center, Room SS024<BR>South Church Street
and Rosedale Avenue<BR>West Chester, PA, 19383<BR>Phone/FAX:
610-738-0437<BR>eMail: fmonson@wcupa.edu<BR>CASI Page and
Scheduling<BR> <A
href="http://darwin.wcupa.edu/CASI/">http://darwin.wcupa.edu/CASI/</A><BR></FONT></P>
<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> Russ Kerschmann
[mailto:rkerschmann@yahoo.com] <BR><B>Sent:</B> Sunday, November 30, 2003 9:27
AM<BR><B>To:</B> histonet@lists.utsouthwestern.edu<BR><B>Subject:</B>
[Histonet] H&E to PAS restaining protocol<BR><BR></FONT></DIV>
<DIV>Hello Histonet,</DIV>
<DIV> </DIV>
<DIV>Can anyone provide a protocol for reprocessing an H&E slide into a
PAS slide? Can the reverse also be performed? How can the
risk of losing the tissue be minimized?</DIV>
<DIV> </DIV>
<DIV>Many thanks, and Happy Holidays.</DIV>
<DIV> </DIV>
<DIV>Russell Kerschmann, M.D.</DIV>
<DIV><A href="mailto:rkerschmann@yahoo.com">rkerschmann@yahoo.com</A></DIV>
<DIV> </DIV>
<P>
<HR SIZE=1>
Do you Yahoo!?<BR><A href="http://antispam.yahoo.com/whatsnewfree">Protect
your identity with Yahoo! Mail AddressGuard</A></BLOCKQUOTE></BODY></HTML>