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. Over fixation can destroy antigens contained within the tissue. Tissue which has dried out can exhibit artifact and nonspecific staining. Incomplete removal of paraffin from tissue sections will result in artifacts.
Poor dehydration causes smudging of the stain.
Thick sections may prevent adequate penetration of primary antibody, producing false reactions or uneven staining over the tissue surface.