Re: [Histonet] restaining of hematoxylin and eosin tissue sections

From:"louise renton"

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.
Some suggestions:

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)

Best regards

Louise Renton
Bone Research Unit
South Africa
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.
Peter Smale
Anatomical Pathology
National Health Laboratory Service
Chris Hani Baragwanath Hospital
Soweto, Johannesburg, South Africa
tel:   +2711 4898711
fax:  +2711 4898717

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