You can never use in a double immunohistochemistry experiment primary antibodies raised in the same species. The primary antibodies must be raised in different species: in this case you can do one step of staining.

I'm sure you knew this.

Good luck with your experiments!

Alessandra Princivalle thank you so much for your reply!

Yes, I know that I must not use two primaries from the same species, but I found a reference in which they do it with these specific two antibodies that basically stain for nuclei and cytoplasm of the same cell type (human cells), so I thought it was possible to do that.

Thank you for your time!

I agree with Alessandra. You need to stain with two primaries from different species and preferably two secondary ABs from different species also. If not, you set yourself up for some harsh criticism from reviewers.

Hi Guilia,

I agree with Alessandra and Sheldon. First a question: Why do you want to stain both antibodies with the same dye (Alexa 488)?

"Traditionally" I would stain both targets with a different dye. If both primaries come from the same species (and it is not possible to switch for a different antibody host in one target) I would recommend either to couple the primaries directly to a different dye (especially if you have enough material from both antibodies) or use to prelabel the primaries with nanobodies attached with different dyes. Here is a link how to do it;

https://nano-tag.com/protocols/multiplexing/

Best regards,

Carsten

Giulia, can you please share the reference you are referring to in your question?

Many thanks

Alessandra Princivalle here the reference: "Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors" Ehrlicha et al., PNAS, 2016.

Many thanks, Giulia.