How do proteolytic enzymes work in IHC?

• A. Cleave specific amino acid sequences within the peptide chain
• B. Cross-link proteins
• C. Hydrolize nucleic acids
• D. Phosphorylate proteins

Answer: (A)

Proteolytic enzymes in IHC work by cleaving peptide bonds within proteins, which helps unmask epitopes that are hidden by fixation. By digesting masking proteins or partially digesting the target protein, these enzymes expose the amino acid sequences that antibodies recognize, enabling better binding and staining. Different enzymes have different preferences—for example, trypsin tends to cut after lysine or arginine, while pronase is broader—so the degree of unmasking can be tuned. This proteolysis helps overcome the masking effect of formalin-induced cross-links and works alongside heat-based antigen retrieval. They do not cause cross-linking (that’s fixation), do not hydrolyze nucleic acids (that would require nucleases), and do not phosphorylate proteins (that’s the job of kinases).