Non-specific binding of Intracellular antibodies - Help!
I’ve run into some issues with my intracellular CyTOF staining. Seems like we get a lot of non-specific binding from our intracellular antibodies such as IL2/Eomes, which we would expect to find in T cells, not in CD33+ cells (see attached photo ). Our extracellular markers as a whole seem to correlate with what we would expect.
Attached is a file with some ViSNE plots showing this non-specific binding. The file has two panels from two experiments:
1) Melanoma sample (Top Panel)
2) PBMC (bottom Panel)
1) ViSNE plots showing expression of 5 intracellular markers and CD33 on Melanoma samples which were stimulated with PMA/Iono for 5 hours and then stained for a variety of extracellular and intracellular markers, many T cells markers.
My staining protocol included:
- A 30min block with 10% Human AB serum
- Extracellular antibody stain
- Fix/Perm using FOXP3 transcription factor kit from eBioscience (2 perm wash steps before intracellular stain).
- intracellular antibody stain (made up in perm wash from FOXp3 kit)
- Two permwashes
- Overnight Iridium in 4% PFA
2) The panel below shows ViSNE plots from an experiment using PBMCs. This experiment was completed using the same antibodies, same staining procedure as the above.
Has anyone else run into this issue? If so, how have you over come this?
Any help would be greatly appreciated!
Daniela