Re: [Histonet] IHC Background Staining

From:"Gayle Callis"

Adding detergent to all buffers and diluents helps, even before the DAB 
step.  This prevents ionic interaction of chromogen to glass slide surface, 
0.05% Tween 20.  Someone can recommend concentration for Triton X-100.   We 
found that drawing a circle around a section was more difficult to blot from 
(the circular edge).  We now draw lines straight across, above and below the 
section, so blotting is done from a tipped slide and from the corner of the 
"square" after adequate rinsing.

Other problems we encountered:

Kits that were older, near expiration
Some kits themselves gave more problems so we switched vendors  OR 
microfiltered the DAB before application (which we disliked doing).
Overdevelopment of the chromogen so the ppt is too heavy.  We now monitor 
all chromogen development with a microscope (the positive control).  This is 
easy to do with manual methods, and taught to me by a master of IHC, Dr. 
Chris van der Loos.   Monitoring allows you to determine an approximate time 
for DAB development, duly noted for that antibody and future use.

Good luck on solving the problem

Gayle M. Callis
Bozeman MT 59715
----- Original Message ----- 
From: "Karla Arrington" 
Sent: Friday, May 09, 2008 5:17 PM
Subject: [Histonet] IHC Background Staining

>I do Immuno's by hand and I am getting background DAB precipitate on the 
>inside circle of my sections.
> What is causing this and how do I get rid of it?
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