Hi Michelle,
2 other possibilities for source of problems are the DAB itself 
and/or the H202. Try a new lot of DAB in case there was a problem 
of storage conditions that you may not be aware of (for instance, 
someone left it out on the bench overnight by mistake, and 
neglected to mention it to you). And I understand (from this list at 
one point, a long time ago), that hydrogen peroxide can go "bad", 
after long term storage, something about forming water molecules. 
I'm sure someone else out there has the proper explanation.
Good luck.
Greg

Date sent:      	Wed, 04 Apr 2001 16:11:08 -0400
From:           	Michelle Peiffer <mlk101@psu.edu>
Subject:        	IHC decreased staining
Forwarded to:   	DOBBIN@acad1.cs.upei.ca
To:             	"histonet@pathology.swmed.edu" <histonet@pathology.swmed.edu>

> 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
> 
> 
> 
> 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
> email:  mlk101@psu.edu
> **************************************************************
>  
>