Which proteolytic enzyme is commonly used for digestion at 37°C in IHC?

• A. Trypsin
• B. Prot K
• C. Pepsin
• D. Pronase

Answer: (A)

Enzymatic digestion in IHC aims to unmask epitopes masked by formalin cross-links by partial proteolysis, and doing this at 37°C helps balance effective exposure of antigens with preservation of tissue structure. Trypsin, a serine protease that cleaves after lysine and arginine residues, is well suited for this purpose in many protocols because it provides reliable epitope exposure without being overly harsh. Digesting at 37°C for a short, controlled time allows antibodies to access their targets more readily while keeping morphology intact, which is why trypsin is commonly used for this purpose. Pepsin requires acidic conditions and can be variably effective depending on the antigen; pronase is a broad, less selective mix that can over-digest; proteinase K is robust and often used under different conditions or for tougher tissues. Hence, trypsin is the most typical enzymatic choice for digestion at 37°C in IHC.