Non-fat milk may mask antigens. Try to reduce the amount of milk in the blocking buffer and antibody solution or substitute it with different blocking reagents.
Use a secondary antibody which was rasied againt the host species of the primary antibody. Use positive controls to ensure that the secondary antibody is working properly.
Use higher concentrations of the antibody. Our protocol provides a guideline for antibody dilutions. Use positive controls to ensure that the primary antibody is working properly.
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