RE: IHC decreased staining

From:"Sebree Linda A." <>


Are you cutting fresh slides each time you stain or are these precut slides
that may be losing antigenicity upon storage?

Linda A. Sebree, HT(ASCP)
University of Wisconsin Hospital & Clinics
IHC/ISH Laboratory
600 Highland Ave.
Madison, WI 53792-2472
FAX: (608)262-7174

-----Original Message-----
From: Michelle Peiffer []
Sent: Wednesday, April 04, 2001 3:11 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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