RE: IHC decreased staining

From:Mary Bryhan <>

To add to what Dave said, most of the antibody vendors will work with you on which antibodies are most vulnerable to antigen loss.  Good luck!

Mary Bryhan HT (ASCP)
Northern Michigan Hospital
Petoskey, Michigan

>>> David Reynolds <> 04/04/01 06:51PM >>>
Hi - I am unaware of how your control slides and tissues are treated, you
may have already thought of this.

There may be some antigenicity lost due to oxidation of your tissue.  If the
control slides are cut well in advance, it would be good to keep them
refrigerated until they are used.  Zymed had a list of antigens that were
particularly vulnerable to this, while I do not have the list in front of me
I do remember that cytokeratins and ER/PR were among them.  I am sure there
will be many other suggestions to follow from the real experts, I am
currently just a dabbler in IHC.

Good luck

David A. Reynolds, HT(ASCP)
6605 Merrill Creek Parkway
Everett, WA  98203

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-----Original Message-----
From: Michelle Peiffer [] 
Sent: Wednesday, April 04, 2001 1:11 PM
Subject: IHC decreased staining

Hi all,

We are have major problems with our IHC and I'm hoping someone out there
may have some insight.  Basically the staining intensity has decreased;
within the same block the DAB is about half as dark as it was 2 months ago,
using the same protocol.  We are using the DAKO autostainer, and DAKO kits
(incidently their tech support is working with me on this too)
I've  tested 6 different antibodies, using 4 different DAKO kits.  I've
stained in the autostainer and by hand.  I've tested the dewaxing agents
(histosolve, xylene, and another xylene substitute) and 2 different methods
of baking the sections onto the slides.  In every case the intensity of DAB
staining is lighter, about 1/2 of the intensity as done previously on the
same blocks.

I've checked the pH of our water used in the buffer.  Strangely our
distilled water pH is 9.1 and our deionized water in pH 9.3.  Though the
buffer itself is 7.5. DAKO is checking to see what effect this may have, it
is definately not what is recommended. 

Any other suggestions?  Thanks for your advice,


Michelle Peiffer
Electron Microscope Facility for the Life Sciences
Penn State University Biotechnology Institute
001 South Frear Lab
University Park PA 16802

phone: 814-865-0212

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