Hello Aly

i think you can incubate the tissue with two primary antibodies at the same time overnight at 4°C . you don't need to stain it separately.

Best Regards

Masuma

I've used up to 4 different antibodies overnight routinely (all from different species) with no issues. Mouse and rat secondary antibodies may have some cross reactivity with primaries--- using a highly cross adsorbed secondary should solve that problem (otherwise, you shouldn't see much cross reactivity between species). Make sure your secondaries do not have overlapping wavelengths of excitation (I regularly use 405, 488, 555, and 647 with no issues on a Zeiss LSM 700)

A double IHC stain can use a combination of red and brown colors. It will be more easy to read if one antibody targets a nuclear Ag and the other a cytoplasmic Ag.

In my case, it works better if incubate the tissue with two primary antibodies it separately and using a humid chamber.

Thanks everyone, I am trying to fine tune the protocol after considering your recommendations.