[Histonet] Confirming IHC staining as "real"

[Morken, Tim - Labvision]

Probably the best is to complex it with the known antigen before putting it
on the slide and then it should be negative. If you don't have the antigen,
then you need a known control and some info on what the ab is supposed to
stain. Negative controls should include 1) no primary or serum (buffer
only), 2) normal serum from host animal of your primary 3) nonsense
antibodies (aren't supposed to stain the target tissue - use ab's from the
same host and preferably same Ig type) and 4) non-target tissues incubated
with your primary(should be negative). 

Tim Morken
Lab Vision / Neomarkers
www.labvision.com
 

-----Original Message-----
From: Smith, Emily [mailto:ESmith <@t> CuraGen.com] 
Sent: Wednesday, March 03, 2004 2:19 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Confirming IHC staining as "real"


Hi All,
What techniques are generally used to confirm immunohistochemistry staining
as real?  If a tissue stains with a labeled primary antibody what follow up
assays and experiments are done to confirm the staining is specific?  
For example, we have tried titering the primary antibody concentration to
see the staining intensity reduced.  We have tried to "compete" the staining
away with unlabeled antibody or with the peptide. Has anyone tried these or
similar approaches?  How were the results? Thanks for your help. ~Emily
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