RE: [Histonet] H&E to PAS restaining protocol
| From: | "Monson, Frederick " |
Message
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@wcupa.edu
CASI Page and
Scheduling
http://darwin.wcupa.edu/CASI/
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.
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