[Histonet] H&E to PAS restaining protocol

[Monson, Frederick]

Dear Russ,
    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%.  
    Now, to the matter of converting an H&E to a PAS.
    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.  
 
    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.
 
        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.
        2.    Soak again to insure complete removal of mountant.  Thus,
two more fresh Xylene rinses for 8 and 16 hours respectively. 
        3.    Bring sections slowly to water.  
                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)
                b.    Any touch of milkiness in sections once they are
in 70% is evidence of residual xylene.  Go back to 100% and re-hydrate.
        4.    Immerse in 1% periodic acid for 10 min at RT.
        5.    My PAS uses the de'Tomasi version of the Schiff's ('S')
reagent (found in Pearse, all editions). Immerse for 10 min at RT,
though I have left for up to 20 min without any observable negative
effect.
        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.
        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.
        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.  
 
NOTE:  I counter with H&E after I process sections or air-dried
mesentery spreads in Gomori's Aldehyde Fuchsin for elastin.
 
        9.  Mount coverglass as required.
 
Hope this helps,
 
Fred Monson (Never known for brevity!)
 

 Frederick C. Monson, PhD
Center for Advanced Scientific Imaging
Mail to Geology
West Chester University of Pennsylvania
Schmucker II Science Center, Room SS024
South Church Street and Rosedale Avenue
West Chester, PA, 19383
Phone/FAX:  610-738-0437
eMail:  fmonson <@t> wcupa.edu
CASI Page and Scheduling
        http://darwin.wcupa.edu/CASI/


	-----Original Message-----
	From: Russ Kerschmann [mailto:rkerschmann <@t> yahoo.com] 
	Sent: Sunday, November 30, 2003 9:27 AM
	To: histonet <@t> lists.utsouthwestern.edu
	Subject: [Histonet] H&E to PAS restaining protocol
	
	
	Hello Histonet,
	 
	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?
	 
	Many thanks, and Happy Holidays.
	 
	Russell Kerschmann, M.D.
	rkerschmann <@t> yahoo.com
	 

	
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