What is a primary purpose of proteolytic digestion in IHC?

• A. Improve penetration of reagents and unmask epitopes
• B. Increase cross-linking of proteins
• C. Remove lipids
• D. Stain nucleic acids

Answer: (A)

Proteolytic digestion in IHC serves to unmask epitopes and improve penetration of reagents. Formalin fixation creates cross-links that can hide antigen sites, making it hard for antibodies to access them. Using proteolytic enzymes (like trypsin or proteinase K) partially digests surrounding proteins, exposing the epitopes and allowing antibodies to bind more effectively. This enhances staining sensitivity, especially for antigens that don’t respond well to heat-based retrieval alone. It’s important to optimize digestion, since too much enzyme can damage tissue structure or destroy epitopes, while too little may not unmask enough for reliable labeling.