Antigen Retrieval Protocol (HIER)

Fixing proteins with formalin creates crosslinks. In the process of crosslinking, antigen in the tissue can be masked, restricting antibody-target binding. The impaired ability of the antibody to access its epitope can result in weak signal or false negative staining. To promote epitope availability and enhance immunogenicity, one of several antigen retrieval methods is used. Proteolytic-Induced Epitope Retrieval (PIER) is an enzymatic method of antigen retrieval which relies on enzymes such as proteinase K, trypsin, or pepsin to unmask antigen. Heat-Induced Epitope Retrieval (HIER) utilizes heat to promote epitope availability. Optimal retrieval conditions depend on the type of tissue, fixation, and antibody, necessitating optimization for each antigen.

Heat-Induced Epitope Retrieval (HIER)

Citrate Buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0):

Citric acid (anhydrous) ------------- 1.92 g

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

Mix to dissolve. Adjust pH to 6.0 w/ 1N NaOH and add 0.5 ml of Tween 20, mix well.

  1. Pre-heat steamer or water bath with staining dish containing Sodium Citrate Buffer or Citrate Buffer until temperature reaches 95-100 degrees Celsius.
  2. Immerse slides in the staining dish.
  3. Place the lid loosely on the staining dish and incubate for 20-40 minutes.
  4. Remove the staining dish to room temperature
  5. Allow the slides to cool for 20 minutes before proceeding with staining procedure.
    Note: Microwave or pressure cooker can be used as alternative heating source to replace steamer or water bath.

Pre-mixed Citrate Buffers

Tris Buffered Saline (TBS) (0.05M TBS, 0.05% Tween 20, pH 9.0):

Tris -------------------------------------- 6.1 g

Sodium Chloride -------------------- 8.8 g

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

Mix to dissolve. Adjust pH to 9.0 w/ HCl. Add 0.5 ml of Tween 20. Mix well.

  1. Pre-heat steamer or water bath with staining dish containing Tris Buffered Saline until temperature reaches 95-100 degrees Celsius.
  2. Immerse slides in the staining dish.
  3. Place the lid loosely on the staining dish and incubate for 20-40 minutes.
  4. Remove the staining dish to room temperature.
  5. Allow the slides to cool for 20 minutes before proceeding with staining procedure.

Note: Microwave or pressure cooker can be used as alternative heating source to replace steamer or water bath.

EDTA Buffer (1mM EDTA, 0.05% Tween 20, pH 8.0):

EDTA (Sigma, Cat# E-5134) ----- 0.37 g

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

Mix to dissolve. Adjust pH to 8.0 using 1N NaOH. Add 0.5 ml of Tween 20. Mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.

  1. Pre-heat steamer or water bath with staining dish containing EDTA buffer until temperature reaches 95-100 degrees Celsius.
  2. Immerse slides in the staining dish.
  3. Place the lid loosely on the staining dish and incubate for 20-40 minutes.
  4. Remove the staining dish to room temperature.
  5. Allow the slides to cool for 20 minutes before proceeding with staining procedure.

Note: This buffer works well for many antibodies, but often results in high background, possibly due to endogenous biotin revealed after antigen retrieval treatment. Due to increased sensitivity, many primary antibody can be highly diluted. We recommend this method for low affinity antibodies or low abundant tissue proteins.

Pre-mixed EDTA Buffers