Hi there, I'm trying to analyse the expression of transcription factors in nuclei using flow cytometry, but am having lots of trouble with non-specific staining. The primary antibodies I am using are purified rabbit polyclonals (which I know are notorious for being sticky), and the second step is goat anti-rabbit-PE. I have titrated the primary and secondary antibodies, and have found that I need to use 1ug/mL of primary antibody/10-6 cells to get decent separation of my antibody and isotype control (decreasing the concentration of my antibody only decreased the intensity of my antibody of interest, and not the isotype control). When I have set the flow cytometer up using an unstained sample, my isotype control sits at around 40 (log scale), and my antibody of interest sits at around 400, and I know that this is sufficient separation, but I would ideally like to reduce my non-specific staining. My secondary step alone does not give any significant level of staining, so I know it's the rabbit polyclonal that's being a pain. I have incubated the nuclei in a blocking buffer containing 4% human serum/5% FCS/0.4%BSA/5% goat serum, but this had no success in reducing the non-specific staining. All washes have been performed using Hanks/0.1%BSA/0.1%azide/0.005%NP-40, and the nuclei have been prepared, fixed and permeabilised using NP-40, paraformaldehyde and tween-20 respectively. Any suggestions on how I can reduce this non-specific staining would be greatly appreciated!! Thanks! Andrea Dewar PhD Student Leukaemia Research Laboratory Department of Haematology Institute of Medical and Veterinary Science Adelaide South Australia 5000 AUSTRALIA
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