[Histonet] IHC double labeling question

[Amy Porter]

I would do them together making sure your end volume holds the same 
concentration of each reagent as you use singely.  It should work fine for 
you and cut down on your time.  You will need to find a generic protein 
block to utilize if your secondaries are made in two different hosts.  If 
your antigens are co-localized you may possibly have a difficult time 
demonstrating them together.  Good luck, Amy
Amy S. Porter, HT (ASCP) QIHC
Investigative HistoPathology Laboratory - Supervisor
2201 Biomedical Physical Sciences Bldg.  Rm #2133
East Lansing, MI  48824-3320
Phone:  (517) 884-5026
Fax:  (517) 432-1368
Email:  portera <@t> msu.edu
Web:  www.humanpathology.msu.edu
----- Original Message ----- 
From: "Nicole Collette" <collette2 <@t> mail.llnl.gov>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Thursday, April 30, 2009 1:16 PM
Subject: [Histonet] IHC double labeling question


> Hi, All,
>
> I am starting to do some IHC on FFPE mouse tissues, and have several 
> antibodies working individually on my tissues (with the same retrieval 
> protocol). The next step is to move on to double labeling, and my generic 
> protocol calls for each label to be done sequentially (primary, secondary, 
> followed by primary, secondary).
>
> My question is, if both of my primary antibodies are raised in different 
> species, and are also different from my host species, can they be done 
> together (mix the primaries for one incubation, mix the secondaries for 
> detection)? It would save a day. I expect to see colocalization, is it 
> better to do both primaries in one incubation so that binding of one 
> doesn't exclude the other? I understand that I will have better control 
> over the post-antibody washes if I do them separately, but is there 
> another reason to do them sequentially if the retrieval is the same? 
> Thanks for the advice!
>
> Sincerely,
> Nicole Collette
> LLNL/UCB
>
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