Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections

Methods Mol Biol. 2010;588:103-19. doi: 10.1007/978-1-59745-324-0_14.


The development of heat-induced antigen (epitope) retrieval (HIER) technologies has led to dramatic improvements in our ability to detect antigens in formalin fixed, archival tissues. Paradoxically, wet heat treatment at temperatures greater than 95 degrees C in appropriate buffer solutions can reconstitute the antigenicity of many proteins that have been rendered nonreactive during the fixation and paraffin embedding process, which heretofore could only be identified in fresh or frozen tissues. The reason for this effect is unclear, but it has been suggested that the vigorous heat treatment partially reverses or disrupts the aldehyde cross-links occurring in proteins during formalin fixation and restores the original conformation of antigenic epitopes. The great success of antigen/epitope retrieval technologies further emphasizes the importance of preanalytical steps in immunohistochemistry. Over the past several years, since this technology was first reported, there have been numerous modifications to the original formulation. It is the purpose of this chapter to discuss the critical issues required for optimal HIER and to provide guidelines for the use of popular HIER buffers and heating devices used for routine immunohistochemical detection.

MeSH terms

  • Animals
  • Antigens / analysis*
  • Antigens, CD34 / analysis
  • Buffers
  • Epitopes / analysis
  • Hot Temperature
  • Humans
  • Immunohistochemistry / methods*
  • Microwaves
  • Palatine Tonsil / chemistry
  • Palatine Tonsil / ultrastructure
  • Paraffin Embedding / methods*
  • Pressure
  • T-Lymphocytes / cytology


  • Antigens
  • Antigens, CD34
  • Buffers
  • Epitopes