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: port...@msu.edu
Web: www.humanpathology.msu.edu
----- Original Message -----
From: "Nicole Collette" <collet...@mail.llnl.gov>
To: <histonet@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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