Hi Lior,

I would need more information to help you.

First question, just to be sure, did you check the specie of your first antibody, and the provider's recommended protocol (permeabilization step, washing buffers, recommended dilution)? Did you try to increase the concentration of your first antibody?

Do you have the reference of your antibody?

Did you do a positive control in your experiment (a well-known antibody with the same secondary that already worked in your hands) to check if the issue was due to the staining process itself?

I don't know the OP9 cell line, but are you sure they are expressing ALP? As it is a mouse cell line, does your antibody is described to target mouse ALP?

If you did all the positive and negative controls, and tried at least 5 concentration of your primary antibody, and if it doesn't work, I would say try another antibody... Usually antibodies for FACS are not expensive, I know that BD Bioscience, Biolegend or Miltenyi are providing good ones.

I hope it helps.

Anne-Sophie