[Histonet] Tumors tumors everywhere

[andreahooper <@t> rocketmail.com]

Did you check to ensure the secondary in the kit is anti-rabbit IgG, with minimal cross to mouse? Ask Biocare directly. What does the background look like? What does your isotype look like? How about your secondary only control?


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-----Original Message-----
From: <sgoebel <@t> mirnarx.com>
Sender: histonet-bounces <@t> lists.utsouthwestern.edu
Date: Thu, 28 Apr 2011 15:16:16 
To: <histonet <@t> lists.utsouthwestern.edu>
Subject: [Histonet] Tumors tumors everywhere

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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