Reducing Background in Immunohistochemistry Methods of Reducing Background Staining in Immunohistochemistry 1. Dilute the primary antibody further, or reduce the antibody incubation time. Staining occurs throughout the section rather than in expected areas of antigen localization. Staining appears to fade, float, or leach off the section during or after substrate reaction.
Color develops immediately after addition of substrate.
Particles of dye are scattered across the section after staining. Reduce substrate staining time. In some instances prolonged staining also inhibits nuclear counterstaining. Some samples contain endogenous peroxidase or phosphatase enzyme. Raise the pH, or increase the ionic strength of diluent buffers.