Antigen Retrieval

Antigen Retrieval

Formaldehyde fixation usually generates methylene bridges which cross-link proteins and therefore mask the epitope of interest. It is essential to unmask the antigen epitopes in order to allow the antibodies to bind, either by heat (Heat Induced Epitope Retrieval: HIER) or enzymatic digestion (Proteolytic Induced Epitope Retrieval: PIER).

HIER

The HIER method can be implemented by microwave, high pressure or water bath. It breaks the methylene bridges and exposes the epitope to allow the antibodies to bind by continuously heating. The following antigen retrieval reagent is required:

  • 0.01 M citrate buffer solution (pH 6.0)
  • 0.01 M PBS buffer (pH7.0)
  • 0.05 M EDTA (pH 8.0)
  • 0.05 M Tris-EDTA (pH 9.0)
  • 0.05 M Tris-HCl (pH 1~12)
  • Microwave Method
    • Place the sample section into a microwaveable vessel where antigen retrieval reagent is present
    • Place the vessel inside a microwave oven
    • Apply microwave radiation to the sample for 5-20 min
  • High Pressure Method
    • Place the sample section into an appropriate vessel where antigen retrieval reagent is present
    • Place the vessel inside a pressure cooker
    • Turn on the cooker and heat the sample until it boils
    • Once boiling starts, turn off the cooker after the sample is allowed to reach full pressure for 1-4 min
  • Water Bath Method
    • Place the sample section into an appropriate vessel where antigen retrieval reagent is present
    • Place the vessel and thermometer inside a water bath chamber
    • Heat the sample to 92℃ in the chamber
    • Remove the sample from the chamber after it is heated at 92℃ for 20-40 min

Notes

  • The temperature and time should be properly controlled for the antigen retrieval methods described above.
  • To avoid original protein structure restoring, do not cool the sample section by taking it out of the buffer solution.
  • The higher the temperature, the shorter the heating time (vice versa).

PIER

Epitope can be exposed by incubation with proteases which can break the methylene bridges. The choice for digestion enzymes depends on the antigenic components. Pepsin and bromelin are used for retrieving antigens in intercellular substance. Other enzymes can be used for intracellular antigen exposure.

Enzyme Working Concentration Digestion Condition
Trypsin 0.05% to 0.1% 37℃ (10 to 40 min)*
Proteinase K 20 µg/mL 37℃ (20 min)
Pepsin 0.40% 37℃ (30 to 180 min)

* The reaction time can be increased for certain worn-out tissues. Fresh trypsin solution should be prepared with pH adjusted to 7.6 and used at 37℃.

  • Frozen
    The most important feature for this type of tissue section is to keep antigen’s immune-competence completely, especially for the cell surface antigen. Both fresh and fixed tissues can be processed as frozen tissues. However, the tissues must be dried (or primary fixed) and stored at low temperature.
  • Paraffin-Embedded
    Paraffin-embedded tissue section is normally sliced by a rotary microtome to give a thickness of 2-7 μm. With proper treatment, the section reveals clear tissue structure and exact antigen location to enable high medical-value pathology researches and retrospective studies. This section type can be stored at 4℃ for long term use.