[Histonet] Tumors tumors everywhere

[Jean-Martin Lapointe]

Hi Sarah,
in a previous job my lab did extensive IHC staining on mouse xenografts of human tumors, like your tissue. We never used mouse monoclonal primaries on this, because of the background issue. Even with a kit designed to supposedly block the non-specific mouse cross-reactivity, the results were not good the few times we tried it.
When using a rabbit poly (or rat mono) as primary, and the appropriate detection system, non-specific background was never a problem. So like Ashley says, your problem is probably with your MACH3 detection system, which seems to pick up mouse antigens. Use a rabbit-specific one, and your problem should go away.
Good luck,

__________________________________
Jean-Martin Lapointe, DMV, MS, dACVP
Vice-President, Pathologie
AccelLAB Inc
1635 Lionel-Bertrand, Boisbriand
Québec, Canada  J7H 1N8
tel:  450-435-9482 ext.247
fax: 450-435-4795
jm.lapointe <@t> accellab.com
 
 


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Message: 10
Date: Mon, 2 May 2011 11:46:29 -0500
From: "Troutman, Kenneth A" <Ashley.Troutman <@t> Vanderbilt.Edu>
Subject: Re: [Histonet] Tumors tumors everywhere
To: "Histonet <@t> lists.utsouthwestern.edu"
	<Histonet <@t> lists.utsouthwestern.edu>
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	<7B310892042DA74CB3590053F424CFE613D63CD2DF <@t> ITS-HCWNEM06.ds.Vanderbilt.edu>
	
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Hi Sarah,

Biocare's MACH3 is designed to work with mouse or rabbit antibodies, so it is likely picking up some mouse antigens:

http://biocare.net/products/detection/mach-3/
MACH 3(tm)

MACH 3 is a two-step, biotin-free detection system which provides excellent specificity, sensitivity and nuclear staining for mouse or rabbit primary antibodies.
I would choose a reagent that recognizes ONLY your rabbit antibody.  Check out Biocare's Starr Trek components (like the Trekkie Rabbit link.  I know the name is goofy... :)).  They should be able to help you.

Good luck.

Ashley Troutman BS, HT(ASCP) QIHC
Immunohistochemistry Supervisor
Vanderbilt University Histopathology
1301 Medical Center Drive TVC 4531
Nashville, TN  37232

Message: 7
Date: Thu, 28 Apr 2011 15:16:16 -0500
From: <sgoebel <@t> mirnarx.com>
Subject: [Histonet] Tumors tumors everywhere
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
      <D957F2A7D21959488C492A2680F9920A224076 <@t> svrexch.asuragen.us>
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So I am staining tumors that were implanted as cells subdermally into
mice.  The cells are human.  I am trying to do Caspase staining on these
tumors.  The primary is an anti-human rabbit polyclonal, and I am using
a polymer (Biocare Mach3) in lieu of the secondary antibody.  The
background is through the roof!!  Could the reason be that the tumor was
grown in a mouse and is having cross reactivity somehow?  What species
antibody should I be using instead?  All my mouse monoclonal antibodies
work perfect on the tissue, it's this stupid rabbit polyclonal!!  I am
blocking endogenous enzymes (peroxidase etc., DAKO), avidin and biotin
(just to see if that would help...it didn't), and protein block (it's
literally an hour worth of blocking!!), developing with DAB (Dako) and
hematoxylin counterstain.  I am so confused as how to get this to work!
Also, it isn't just this particular antibody it is any rabbit polyclonal
I have tried.  Could it be the polymer?  It is the one that Biocare
suggested?  HELP!!

Thanks in advance =)



Sarah Goebel, BA, HT(ASCP)

Histotechnologist

Mirna Therapeutics

2150 Woodward Street

Suite 100

Austin, Texas  78744

(512)901-0900 ext. 6912



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