Hydrochloric Acid (HCl) 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 HCl 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:  


Hydrochloric Acid (HCl) Solution (2N in Distilled Water, pH 0.6-0.9):

      10N (concentrated) HCl ----------------- 20 ml

      Distilled water ---------------------------- 80 ml

      Mix well and pH should be around 0.6-0.9

      Incubate sections for 10-20 minutes at room temperature.



  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. Incubate sections with 2N HCl solution for 10-20 minutes (optimal incubation time should be determined by user).
  4. Rinse sections in PBS Tween 20 for 2x2 min.
  5. Block sections with for 30 minutes.
  6. Perform avidin/biotin blocking if necessary.
  7. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C.
  8. Rinse sections with PBS Tween 20 for 2x2 min.
  9. Block sections with peroxidase blocking solution for 10 minutes.
  10. Rinse with PBS Tween 20 for 3x2 min.
  11. Proceed to standard immunohistochemistry protocol.

Note: This method works well with BrdU and beta amyloid antibody staining.