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Antigen Retrieval Protocols – Universal Antigen Retrieval Method
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Antigen Retrieval Protocols – Universal Antigen Retrieval Method

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Description:Formalin is a commonly used fixative for tissue preservation in histopathology labs. A major adverse effect of this fixative is the concealing of tissue antigens by protein cross-linking through formaldehyde reaction with the protein amino groups. Citraconylation by citraconic anhydride provides an easy method for the reversible blocking of protein amino groups. The method provides efficient AR for successful immunostaining of a wide variety of antigens under an optimized condition.

Solutions and Reagents:  

0.05% Citraconic Anhydride Buffer (pH 7.4):

  • Citraconic anhydride ------------------ 0.5 g
  • Distilled water --------------------- --- 1000 ml
  • Mix to dissolve. Adjust pH to 7.4


  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 Citraconic Anhydride 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: Since citraconic anhydride is considered a toxic agent, it is necessary to use a hood and to prevent contact with skin and eyes when performing the AR procedure, although the concentration needed was very low.


1. Namimatsu S, Ghazizadeh M and Sugisaki Y (2005) Reversing the effects of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method. J Histochem Cytochem. 53(1):3-11. PubMed Abstract

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