I'm conducting immunofluorescent experiments with white blood cells (PBMCs that isolated with Ficoll) and various cell lines. ER antibody (EPR4097 clone) and PR antibody (SP2 clone) are giving fluorescent signal on the WBCs. I worked with different blood samples and repeated this experiment 3 times, but non-specific binding has occured all the time.
My protocol: Fixation with 4% formaldehyde for 10 min, permeabilization with 0.1% Triton-X-100 for 5-10 min, blocking with 1%BSA for 30 min (I tried 1% BSA+10%FBS for 1 h and it did not work either) and antibody incubation with 1% BSA in PBS for 1 h at room temperature. I did not use secondary antibodies, primary antibodies have Alexa Fluor 488 conjugate. I've worked with this dye before so the problem isn't with our microscope or fluorescent filters.
Do you have any recommendations?
It is quite possible that your staining antibodies are binding to the PBMC via Fc receptors. You can block that binding with either purified antibodies of the same species [purified mouse IgG if your staining antibody is a mouse monoclonal] or using an FcR blocking antibody. Miltenyi sells an FcR block reagent that I have used and it works quite well.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies