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Blocking

One important way to minimize non-specific staining is by the use of a so-called blocking reagent. A blocking reagent contains a high concentration of immunoglobulin that will bind to the Fc-receptors on cells like monocytes, thereby blocking the non-specific binding of the staining antibody reagents to these receptors. The blocking reagent should be immunoglobulin from the species whose cells you are staining. For human cells, use the g-globulin fraction from human serum at a final concentration of 4 mg/ml. Alternatively, one can purchase specific antibodies directed against the Fc receptors of the cells in question; care needs to be taken here as there are different classes of Fc receptors and, in addition, not all antibodies against Fc receptors block the receptor binding site.


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