Table 2.

Troubleshooting Guide for Immunohistochemistry assay

Problem	Possible Cause	Solution
High background staining	Secondary antibody cross-reactivity or nonspecific binding is present	Increase the serum concentration, alternatively, decrease the concentration of the secondary antibody.
	Issues with the primary antibody, which may show an affinity for similar epitopes on non-target antigens	Reduce the concentration of the primary antibody, adjust the concentration of triton-x in the blocking solution, or use a different primary antibody.
Weak target staining	Poor primary antibody potency (due to protein degradation/denaturation caused by long-term storage, repetitive freeze/thaw cycles, microbial contamination, changes in pH, etc.	Ensure that the antibody is correctly stored (at −20C or 4C) according to the manufacturer’s instructions in small aliquots, use sterile pipette tips and tubes.