[Histonet] IHC double labeling question

[Nicole Collette]

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