[Histonet] Rabbit-onRodent HRP Polymer

[david.l.krull <@t> gsk.com]

Hi Jen,

The rabbit-on-rodent HRP ploymer is ready-to-use.  In my protocol I depar 
and hydrate to wash buffer then block for endogenous peroxidase for 10 
minutes using Dako's DEEB followed by wash buffer rinses then block for 15 
minutes in Rodent Block M (Biocare).  No need to block for biotin since no 
streptavidin is used in the detection.  I suggest that you first try the 
block you have.  Apply rabbit primary antibody for 30 minutes, rinse in 
wash buffer, then apply the rodent-on-rabbit HRP polymer for 15 to 30 
minutes.  Rinse in wash buffer again then apply your DAB chromagen for 5 
minutes.  Rinse well in DI water then stain with hematoxylin.  I run all 
of my IHC on a Lieca Bond Max using the Biocare reagents and they work 
great.  This protocol intended for general guidance; the exact conditions 
will need to be worked out in your lab.

Hope this helps.

Dave

 
Jen wrote:

Message: 11
Date: Thu, 15 Oct 2009 17:36:41 -0700
From: "Jennifer Campbell" <jcampbell <@t> vdxpathology.com>
Subject: [Histonet] Rabbit-on-Rodent HRP polymer
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
 <5658CBDB9EAE6545ABE50D2563D81BF8181FB5 <@t> VDXSERVER01.vdxpathology.local>
 
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Hi All,
 
  I recently purchased a rabbit-on-rodent HRP polymer from Biocare.  I 
will be using this on mouse tissue, using a rabbit polyclonal CD3 as my 
primary.  I just wanted to clarify a couple things about the protocol 
before I try it out tomorrow and was wondering if anyone could offer some 
assistance.  First of all, I should aske if anyone has a protocol for 
this?  What I really would like to make sure is that I will not have to 
use a label after the rabbit-on-rodent secondary ab.  I believe that I 
will just have to apply my blocking agent, avidin/biotin blocks, primary 
antibody, secondary anitbody, followed by chromogen, right?  My blocking 
agent is made up of casein and some other proprietary agents in a 
phosphate buffer (it is non-serum) so I was told that should work fine. 
Does this sound correct?  Thanks in advance!  I haven't been able to get 
through to tech support all day, so hopefully you will have some answers 
for me!
 
Jen