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

From:"Peter Smale"

George Cole
The slide was left out exposed to sunlight for a prolonged period, probably several weeks and yes all our slides are coverslipped using the same coverslipping tape.
.....................................................
Peter Smale
Anatomical Pathology
National Health Laboratory Service
Chris Hani Baragwanath Hospital
Soweto, Johannesburg
tel:   +2711 4898711
fax:  +2711 4898717
e-mail: peter.smale@nhls.ac.za
 
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----- Original Message -----
From: George Cole
To: 'Peter Smale'
Sent: Tuesday, November 25, 2003 10:43 AM
Subject: RE: [Histonet] restaining of hematoxylin and eosin tissue sections

Peter,

No clear answers from me, but one vague guess----  What caused the H & E to fade? H & Es seem to hold up pretty well. Did a lot of people look at the slide for prolonged periods under bright light? If not,  I would consider the covering method----thats the only difference that I can see between that H & E and a million other unfaded H & Es. It doesnt seem likely that it involved the reagents you used to remove the cover and the residual of the covering, because the fading had already taken place. Do you have any other H & Es covered the same way?  How are they? I hope you solve your problem.

georgecole@ev1.net    .     

-----Original Message-----
From: histonet-admin@lists.utsouthwestern.edu [mailto:histonet-admin@lists.utsouthwestern.edu] On Behalf Of Peter Smale
Sent:
Tuesday, November 25, 2003 12:17 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] restaining of hematoxylin and eosin tissue sections
Importance: High

 

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
e-mail: peter.smale@nhls.ac.za

 

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