Hydrochloric Acid Antigen Retrieval Protocol


Hydrochloric acid antigen retrieval is used with paraffin embedded, formalin or paraformaldehyde-fixed tissues. When tissues are immersed in cross-linking agents, such as from paraformaldehyde, these agents will covalently cross-linked proteins, resulting a reduction in the available epitopes for antibody binding. The result is weak or negligible levels of protein detection. Consequently, the use of a relatively strong hydrochloric acid solution has been shown to greatly improve binding for some antibodies. However, not all antibodies respond equally well to a low pH solution treatment, and several antigen retrieval condition should be tested when optimizing stainng conditions.


Hydrochloric acid solution, 2N

  • Prepare the solution by adding 20 ml of 10N (concentrated) HCl to 80 ml of distilled water. Be sure to mix the solution well; the pH should be in the range of 0.5-1.0.


  1. Deparaffinize and rehydrate tissue sections.
  2. Incubate sections using the 2N hydrochloric acid solution for 10 to 20 minutes at room temperature. A range of times (suggestion 10, 15, and 20 minutes) incubation time should be tested to determine an optimal antigen retrieval time.
  3. Rinse sections in two changes of a buffered solution, such as PBS or TBS, in order to neutralize acidity.
  4. Proceed with the tissue blocking for immunohistochemistry.


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