Tris-EDTA Antigen Retrieval Protocol


A Tris-EDTA buffer antigen retrieval solution is used with paraffin embedded, formalin or paraformaldehyde-fixed  tissues [1].  When tissues are fixed in cross-linking agents such as paraformaldehyde, these agents will covalently cross-linked proteins, resulting a reduction in the available epitopes for antibody binding. EDTA buffer is used to improve staining with low abundance epitopes and with antibodies that have weak affinity; it appears to enhance staining for many antibodies, although background staining is often increased.


Tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH 8.0)

  • Mix 1.21 g Tris Base, 0.37 g of EDTA in 1000 ml of distilled water. Adjust the pH to 9.0 with 1N sodium hydroxide and then add 0.5 ml of Tween 20.
  • Store at room temperature for up to 3 months; for extended storage, store at 4oC.


  1. Deparaffinize and rehydrate tissue sections.
  2. Preheat a steam or a water bath containing a staining dish filled with  antigen retrieval buffer to 95-100oC.
  3. Immerse slides in the staining staining dish and incubate for 20-40 minutes; time is empirically determined with a suggestion for 20, 30 and 40 minutes as initial starting conditions.
  4. Remove the staining dish and allow slides to cool for 30 minutes before immersing in wash buffer in the next step. The cooling process prevents tissue from falling off the slide.
  5. Rinse sections in two changes of a buffered solution, such as PBS or TBS, in order to eliminate remaining antigen retrieval solution.
  6. Proceed with tissue blocking for immunohistochemistry.


  1. Pileri SA, Roncador G, Ceccarelli C, et al (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol. 183(1):116-23. PubMed Abstract


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