Just get different species antibodies. No reviewer worth their salt will trust that there is no cross reactivity in sequential staining. I certainly wouldn't. If your primaries are different isotypes, you can get specific secondaries though, even within the same species.

Hi Syed,

In theory you can do that, it is called sequential immunohystochemistry. It doesn't yield results as good as the simultaneous incubation, but it should work, although you will have to optimize the concentrations and incubations of each antoibody and this can be a time-consuming process. You will also need three different fluorochrome labeled anti-rabbit secondary Abs.

See the link below, this is a guide of Abcam for sequential IHC. This question has been posted previously in RG forums so I also send you the link to this discussion.

http://www.abcam.com/protocols/immunocytochemistry-immunofluorescence-protocol

https://www.researchgate.net/post/How_can_I_perform_double_immunofluorescence_staining_using_two_monoclonal_antibodies_in_the_same_sample_preparation2

Hope it helps,

cheers

 thanks ever so much

Just get different species antibodies. No reviewer worth their salt will trust that there is no cross reactivity in sequential staining. I certainly wouldn't. If your primaries are different isotypes, you can get specific secondaries though, even within the same species.

I agree with Niels. It is more reliable to apply primaries from different species.

There is no need to do old-fashioned stuff like adjacent sections and DAB, this isn't the 1980s any more. Just be sensible, get different species primaries, choose secondaries that are nice and far apart (488 and 647 are my favourite pair) and get on with it. Nobody is going to argue with that.

thanks all

Primaries from different species are expected to work better