Tris Buffer 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 Tris 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:

 

Tris-Buffer (10mM Tris Base, 0.05% Tween 20, pH 10):

      Tris Base -------------------------------- 1.21 g

      Distilled water -------------------------- 1000 ml

      Mix to dissolve. Adjust pH to 10 using 1N NaOH and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 ºC for longer storage.

      Note: It is sometime occurred with tissue damage or sections detached from slides when using this method. It is probably due to high pH value.

 

Procedure:

  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 Tris 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.

 

 

 

References:

 

1. Taylor CR, Shi SR, Chen C, Young L, Yang C, Cote RJ (1996) Comparative study of antigen retrieval heating methods: microwave, microwave and pressure cooker, autoclave, and steamer. Biotech Histochem. 71(5):263-70. PubMed Abstract