As mentioned by William, it's the best to have primary antibodies raised in different species, and so avoiding the cross reactivity. But if your primaries are of different isotypes, and your secondary is specifically bind to the one of them - in this case (in addition to the controls you have) you may be able to go on for this experiment.

I agree with the opinion of William and Ibrahim.

Can you use the Rat anti-mouse PD1 from Biolegend corp. or from other source like Cell Signaling?

Thank you for your answers. But if I use antibodies from rat, wouldn't there be a non-specific binding since I'm testing on mouse cell line?

use secondaries that are preadsorbed for mouse. However you can prelabel your antibodies and there are kits available that work quite well.

The APEX labeling technologies is suitable for small amounts of antibodies in complex solutions with stabilizing proteins like albumin or gelantin.

http://www.lifetechnologies.com/de/en/home/life-science/cell-analysis/labeling-chemistry/protein-and-antibody-chemical-labeling/antibody-protein-labeling-kits/apex-antibody-labeling-kits.html

The you can use two different colors without having the species problem.

If you have larger and pure amounts of the antibodies you can directly buy reactive dyes and label yourself a higher amount. This will probably be cheaper.

Good luck,

Henrik

Your primary antibodies must be from two different species unless (1)you have known the two primary antibodies very well. e.g. there is NO unspecific binding or at least, the unspecific binding won't interfere with the signal of another primary antibody. (2)And the molecular weight of the two proteins of interest are different enough.

For the secondary antibodies, actually, you only need to use one of them. The potential competition between the two anti-rabbit antibodies might give you weaker signal. However, you might use two anti-rabbit antibodies conjugated with the same dye for enhanced signal.