Protocol for Heat-Induced Epitope Retrieval (HIER)

Antigen retrieval can reveal epitopes masked during the preparation of tissues for staining. The protocol below describes a technique used by R&D Systems and can be used on both cryostat and paraffin-embedded sections. The protocol can be used with R&D Systems Acidic, Basic, or Universal Antigen Retrieval Reagents. Pretreatment with these solutions may induce a dramatic enhancement of immunoreactivity. However, they also have the potential to affect tissue morphology. The optimal method of antigen retrieval must be determined experimentally.

Please read the protocol in its entirety before starting.

Reagents Required for Heat-induced Epitope Retrieval

  • 10X Antigen Retrieval Solution:
    Antigen Retrieval Reagent-Basic (Catalog # CTS013) or
    Antigen Retrieval Reagent-Acidic (Catalog # CTS014) or
    Antigen Retrieval Reagent-Universal (Catalog # CTS015)
  • Deionized H2O
  • 1X PBS: 0.145 M NaCl, 0.0027 M KCl, 0.0081 M Na2HPO4, 0.0015 M KH2PO4, pH 7.4


  • Polypropylene Coplin staining jar (or equivalent)
  • Water bath at 92-95 °C


  1. Make working dilutions by mixing 1 part of 10X Antigen Retrieval concentrate with 9 parts of deionized water.
  2. Preheat retrieval solution to 92-95 °C. This may be achieved by placing a polypropylene Coplin staining jar filled with retrieval solution into a water bath.<
    Note: Heating may cause cracking of glass staining dishes.
  3. Immerse slides into preheated retrieval solution for 2-10 minutes.
    Note: Since the effect of antigen retrieval reagents depends on their temperature (90-100 °C) and incubation time (up to 30 minutes), optimal conditions should be determined by the individual investigator.
    Note: Cryostat sections are more sensitive to damage by retrieval solution than paraffin-embedded tissues. To avoid tissue damage, it may be necessary to shorten the incubation time to 2-5 minutes.
  4. After the incubation is finished, remove the Coplin jar with retrieval solution and slides from the water bath, and let it cool to room temperature.
  5. Gently rinse the slides with deionized water and then with PBS.
    Note: Because tissues may be loosened after the retrieval procedure, avoid vigorous rinsing to prevent detachment from the slides.
  6. Tissues are now ready for blocking and primary antibody incubation.

Optimization of HIER conditions

The table below depicts a typical experimental set-up to determine optimum HIER incubation time and pH. Results should be compared to a tenth slide, with no HIER. A similar approach can be employed to optimize incubation temperature.

Time Antigen Retrieval Solution pH
Acidic Neutral Basic
1 minute Slide #1 Slide #2 Slide #3
5 minutes Slide #4 Slide #5 Slide #6
10 minutes Slide #7 Slide #8 Slide #9

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