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Pepsin Antigen Retrieval Protocol
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Pepsin Antigen Retrieval Protocol

Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The pepsin based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies.

Solutions and Reagents:  

Pepsin Stock Solution (1% in 10mM HCl):

  • Pepsin ----------------------------------- 100 mg
  • 10mM HCl (pH 2.0) --------------------- 10 ml
  • Mix to dissolve. Store at -20 ºC

Pepsin Working Solution (0.5% in 5mM HCl):

  • Pepsin Stock Solution (0.5%) ------------ 1 ml
  • Distilled water ---------------------------- 1 ml
  • Mix well.


  1. Deparaffinize sections in 2 changes of xylene, 5 minutes each.
  2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each.
  3. Rinse in distilled water.
  4. Cover sections with pepsin working solution and incubate for 10-20 minutes at 37 °C in humidified chamber (optimal incubation time may vary depending on tissue type and degree of fixation, and should be determined by user).
  5. Allow sections to cool at room temperature for 10 minutes.
  6. Rinse sections in PBS Tween 20 for 2x2 min.
  7. Block sections with for 30 minutes.
  8. Perform avidin/biotin blocking if necessary.
  9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C.
  10. Rinse sections with PBS Tween 20 for 2x2 min.
  11. Block sections with peroxidase blocking solution for 10 minutes.
  12. Rinse with PBS Tween 20 for 3x2 min.
  13. Proceed to standard immunohistochemistry protocol.

Note: This method tends to produce tissue damage so incubation time is import factor to consider when using this method. Select appropriate incubation time for a specific application.

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