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
shive...@umn.edu
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- Original Message -
From: "Nancy Herman"
To:
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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