I have been testing antibodies against nuclear factor 1 (NF1) transcription factors by flow cytometry on splenocytes.

This antibody is initially made for western blotting or IP but we biotinylated it and are using it by performing 2 step intracellular staining with streptavidin PE.

Link to the antibody I'm using: https://www.bethyl.com/product/A303-565A?referrer=search

As for my controls, my FMO is completely negative, and my FMO + secondary antibody (strep-PE) is also negative. However, the isotope control gives a high signal, almost as high as my stained sample. The isotope control I'm using is from Sigma and is host-matched (Rabbit), isotope matched (full IgG) and was biotinylated at the same time as my NF1 antibody.

My protocol is basically Fc blocking, surface staining, Fix/Perm with BD Pharmingen transcription buffer set, Perm/Wash, Intracellular staining with anti-NF1B in perm/wash buffer and secondary staining with strep-PE in perm/wash buffer. I did take a break and left my cells over night in perm/wash buffer after fixation and before intracellular staining. People from my lab told me they had done it before without any problem.

Has anyone ever had a similar problem with isotype controls?