I want to do an immunolocalization of two protein in an embryo. They localize in the same type of cells. What do you think about double immuno? Is it better to coincubate or not? This question is not only for primary but also for secondary antibodies incubation.
I have experience with double immunofluorescence for the same type of cells, and I can tell you that both sequential and simultaneous incubation work fine as long as your primary antibodies raised in different species (usually mouse and rabbit). We use a mix of secondary antibodies raised in the same species (usually goat) against the primary antibodies. You can find more information on abcam webpage.
Double immunofluorescence works well with primary antibodies raised in different species and then mixed secondary antibodies. If you are doing double IHC with DAB or other chromogens, you could try using serial sections stained for different proteins and comparing them for co-localisation, or choose chromogens with contrasting colours and do the staining sequentially.
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