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Citrate-EDTA Antigen Retrieval Protocol
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Citrate-EDTA 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 citrate-EDTA 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:  

Citrate-EDTA Buffer (10mM Citric Acid, 2mM EDTA, 0.05% Tween 20, pH 6.2):

  • Citric acid (anhydrous)  ----------------- 1.92 g
  • EDTA (Sigma, Cat# E-5134) ------------- 0.74 g
  • Distilled water -------------------------- 1000 ml

Mix to dissolve. Adjust pH to 6.2 and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.


  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. Then rinse in distilled water.
  3. Pre-heat steamer or water bath with staining dish containing Citrate-EDTA buffer until temperature reaches 95-100 °C.
  4. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation time should be determined by user).
  5. Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 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: Microwave or pressure cooker can be used as alternative heating source to replace steamer or water bath.


1. Sheriffs IN, Rampling D, Smith VV (2001) Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy. J Clin Pathol. 54(7):517-20. PubMed Abstract

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