Which enzymes are used for intracellular antigen exposure in immunohistochemistry?

• A. Trypsin 0.05% to 0.1% 37°C (10 to 40 min)
• B. Proteinase K 20 µg/mL 37°C (20 min)
• C. Pepsin 0.40% 37°C (30 to 180 min)
• D. All of the above

Answer: (D)

Enzymatic antigen retrieval uses proteolytic digestion to unmask epitopes that are hidden by formalin cross-links, making intracellular targets accessible to antibody binding. Trypsin, proteinase K, and pepsin are all proteases commonly used for this purpose, each under specific conditions to suit different antigens and tissues. Trypsin digests proteins at Lys-Arg sites and is effective for exposing cytoplasmic and some membrane-associated antigens. Proteinase K provides broad proteolysis and is particularly useful when more robust or nuclear intracellular epitopes need exposure. Pepsin, active in acidic conditions, can reveal certain intracellular epitopes that respond to gentler but acidic proteolysis. Because each enzyme can be employed for intracellular antigen exposure depending on the antigen, tissue, and fixation, using all of them covers the range of scenarios encountered in practice.