[Histonet] Mouse lung IHC false positive/background issue

[Patsy Ruegg]

if any part of your detection is something anti mouse you will get binding to all mouse tissue Ig not just the Ig associated with the antibody u are using.  What is the host species of the antibody being used?  Most labeled polymer detection systems use a link step which is usually rab anti mouse for mouse antibodies to link the rab portion to the labeled polymer 3rd step which is usually goat anti rab if you are using a rab antibody on mouse tissue u can skip the link step and go directly to the goat anti rab labeled polymer, avoiding anti mouse reagents. 

From: NMargaryan <@t> luriechildrens.org
To: lu_ze <@t> sbcglobal.net; histonet <@t> lists.utsouthwestern.edu
Date: Fri, 11 Oct 2013 20:01:55 +0000
Subject: RE: [Histonet] Mouse lung IHC false positive/background issue
CC: rebeliza <@t> gmail.com

I have same problem for other Abs using in on mouse lung tissues... My secondary and tertiary have no problems on other tissues for negative controls as well....
 
Naira
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Lu Ze
Sent: Friday, October 11, 2013 2:38 PM
To: histonet <@t> lists.utsouthwestern.edu
Cc: rebeliza <@t> gmail.com
Subject: [Histonet] Mouse lung IHC false positive/background issue
 
Hello everyone,
 
I forwarded message from my colleague, Rebecca (see below) to see if any one with IHC experience on mouse lung tissue have suggestion.
 
 
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I'm having trouble with my IHC. I am staining for human MTDH (specific for human) on mouse tumor and lung sections from my xenograft mouse model. All of my tumors stained positive, but not the lung sections (which could possibly be the results). My real problem is that my negative control normal lung section stained positive for MTDH. I saw positive staining on the tissue near the outside and in the bronchioles.
 
I know the problem is not the secondary antibody because I did 2 slides (normal lung and previously positive tumor) without primary ab and they showed no positive staining.
 
My antibody is a rabbit monoclonal antibody at 1:200 for 1 hour. I'm blocking with 1% BSA in TBST for 1 hour. I'm using DAB substrate kit for developing.
 
What would you recommend?
 
Thank you,
 
Ze
 
================================
Ze Lu, Ph.D.
Optimum Therapeutics, LLC
9363 Towne Centre Dr., Suite 110
San Diego, CA 92121
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