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. Apply blocking protein prior to the application of primary antibody. The blocking protein should be of the same species used in the link or enzyme labeled antibody diluent, and should not be used in the primary antibody diluent. The addition of ethylene glycol, Tween 20 or other detergent to the antibody diluent and washes will help remove unbound antibodies and artifacts which contribute to non-specific binding, as well as remove excess dye from the section. Try an alternative enzyme system, such as alkaline phosphatase, horseradish peroxidase, or b -galactosidase. 8.