I'm carrying murine bone marrow-derived dendritic cells cultures. I was having trouble with the signal of the isotype control for anti-CD11c+ PECy5, because the signal was so intense that when I had to set the gate for positive cells, it resulted in a very low percentage in the test group. So I changed the strategy and increased the concentration of Fetal Bovine Serum from 5% to 10% on the blocking solution (PBS) for the isotype and the problem was solved. My question is, should I use the same concentration (10%) of FBS on the blocking solution of the test monoclonal Ab as well? I used FBS 5% as usual and it was okay, but I want to know if it's methodologically correct to use different concentrations for the isotype control and test Ab.
Hi,
I agree with Seppe. Always do exactly the same with your isotype control than with your antibody of interest.
That way any chnage you will see with your CD11c antibody for instance will be true.
Alex
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