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IHC Troubleshooting Antigen Retrieval

Antigen retrieval (AR) is the process of breaking protein cross-links that mask antigens in formalin-fixed tissue sections. Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, giving weak or false negative staining. Antigen retrieval breaks the protein cross-links, enhancing staining intensity by unmasking the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections.
Problems with the antigen retrieval step of IHC can result in weak signal. Troubleshoot your IHC experiment with these tips:

  • Evaporation can cause the slide to dry out during AR; increase the volume of antigen retrieval solution in order to cover the slides more completely
  • Run a control experiment to determine the optimal amount of time for antigen retrieval. Use slides of the same tissue section and run the retrieval for 5, 10, 15, 20, 25 and 30 minutes before staining to evaluate optimal antigen retrieval time for the antibody being used.
  • Allow the slides to cool completely before handling. This allows the antigenic sites to re-form after being exposed to high temperature.

Keywords: Antigen retrieval, Immunohistochemistry, IHC Troubleshooting, IHC