[Histonet] GFAP IHC

[Jan Shivers]

Nancy,

I don't use the ready-to-use GFAP from Dako, but I do use the concentrated 
form of GFAP (it is also a rabbit anti-cow GFAP.)  It's worked successfully 
in dog, cat, pig, cow, horse, goat, sheep, deer, eagle, mouse, rat, gerbil, 
guinea pig, hamster, monkey, ferret, and kangaroo with no background 
problems whatsoever.

The EnVision+HRP Rabbit will only bind to the rabbit IgG, not the cow IgG, 
so that is not the source of your background.  I would suspect that if 
you're getting brown staining on the slide itself, your antibody is much 
much too concentrated.  My working concentration calculates out to be 4 
ug/ml of Ab.  You might see if your ready-to-use preparation has a 
concentration listed, and then work from there to approximate a 4 ug/ml 
concentration with diluting it out.

If that doesn't work... buy the concentrated stock form and optimize it for 
your tissues.  In my lab, it requires absolutely no tissue pretreatment, and 
the stain is always strong, clear and clean.  Good Luck!

Best regards,
Jan Shivers
Senior Scientist
Pathology Teaching Program
Histology/IHC/EM Section Head
University of Minnesota
Veterinary Diagnostic Laboratory
1333 Gortner Ave.
St. Paul, MN  55108
612-624-7297
shive003 <@t> umn.edu

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----- Original Message ----- 
From: "Nancy Herman" <Nancy.Herman <@t> inspection.gc.ca>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Wednesday, October 28, 2009 3:19 PM
Subject: [Histonet] GFAP IHC


> We are using DAKO's ready-to-use GFAP antibody on our bovine tissue and 
> using Envision+ HRP Rabbit detection system.  When using this antibody we 
> see a brown color on the slide itself.  The staining was intense and quite 
> uneven.  We have diluted the anitbody to 1:10 but there is still the brown 
> slide.  The staining is not quite as uneven but appears more washed out. 
> We don't see this with any of our other antibodies we are using and our no 
> primary controls for GFAP are clean.  Any advice or explanations for the 
> slides picking up the brown color or improving GFAP IHC on bovine tissue?
> Thanks
>
> Nancy Herman
> CFIA Lethbridge Laboratory
> Lethbridge, Alberta


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