[Histonet] double stain

[Orr, Rebecca]

Rachel,
I have encountered similar issues in my journey through doublestaining.

>From what I know of Triton X, it's mainly a surfactant.  I'm not sure
of the pH.  There are solutions on the market that most likely have some
type of surfactant in them, hence all the soapy bubbles in the rinsing.
What solutions have you tried? 

What I have been successful with in this type scenario is trying a
combination of either a lighter (less harsh HEIR) and then either  a
diluted enzyme or less time in the enzyme.  Can you alter the
concentration of the antibodies?  One using the triton might need higher
concentration, or more time.
When you say sequentially, are you taking one ab all the way to the
chromogen then starting the second one? I would imagine this is your
protocol, but that would be good to know.

 Becky Orr CLA,HT(ASCP)
IHC Lead 
Evanston Northwestern Healthcare
847-570-2771
 

 

Message: 28
Date: Fri, 17 Mar 2006 07:42:38 -0500
From: "Emerson, Rachael" <Rachael_Emerson <@t> URMC.Rochester.edu>
Subject: [Histonet] Double stain ?
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <C04018EE.23AB%rachael_emerson <@t> urmc.rochester.edu>
Content-Type: text/plain;	charset="iso-8859-1"

Hello. I am attempting to do a sequential double stain with 2 different
antibodies.
Unfortunately, one requires using Triton-X 100 for antigen retrieval and
the
other requires using trypsin.
I've run trials using several different antigen retrieval methods and
these
are the only ones that work.
I'm afraid that performing 2 different retrieval steps will cause the
stain
not to work. I would really appreciate any thoughts or suggestions.
I am working with paraformaldehyde fixed mouse embryos.


Thanks 
Rachael Emerson





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