Re: [Histonet] restaining of hematoxylin and eosin tissue sections
Did you hand stain or machine stain the slide? If it was automated, perhaps
the surface tension on the slide was altered in some way and the reagents
did not have the opportunity to "stick" onto the section., perhaps the
graded alcohols are contaminated with xylene.
a) Rinse slide in fresh abs alc
b) Try restaining by hand to monitor progress
c) Use the counterstain with the acetic acid omitted. This might be fading
your nuclear staining. ( I seem to recall a pernicious habit of "dosing" the
eosin with acetic acid as the week wore on - after a couple of days it was
sufficient to wash out the haem)
Bone Research Unit
Tel & fax +27 11 717 2298
"Time flies like an arrow, fruit flies like a banana"
----Original Message Follows----
From: "Peter Smale"
Reply-To: "Peter Smale"
Subject: [Histonet] restaining of hematoxylin and eosin tissue sections
Date: Tue, 25 Nov 2003 10:17:24 +0200
I wonder if someone can assist me in solving this problem.I retrieved a
hematoxylin and eosin/phloxine stained slide from 1998 and discovered it was
faded.The tissue section was mounted in a coverslipping machine that uses
coverslipping tape composed of cellulose triacetate and is coated on one
side with resinous mountant.This is the procedure I followed to
decoverslip,decolorise and restain: I placed the slide in acetone for 7
minutes to remove the coverslip and then put the slide in xylene for 6 hours
to remove any residual resinous mountant.I took the slide through graded
ethanols to water after which I decolorised with acid/alcohol for one
minute, took the slide back to water and restained the nuclei with Gill's
Hematoxylin ( for 5 min. ) and counterstained with a mixture of 2% eosin y
and 1% phloxine B with 4 drops of conc. glacial acetic acid added ( for 3
min. ).The nuclear staining turned out pale and the counter stain was patchy
almost as if something was blocking the tissue's ability to retain the dyes
I cannot figure out why the tissue is not staining with the same intensity
as it did in 1998.Please keep in mind the slide was left out in natural
light for a while which resulted in it becoming faded.
National Health Laboratory Service
Chris Hani Baragwanath Hospital
Soweto, Johannesburg, South Africa
tel: +2711 4898711
fax: +2711 4898717
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