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 # VCTS021) or
  Antigen Retrieval Reagent-Acidic (Catalog # VCTS022) or
  Antigen Retrieval Reagent-Universal (Catalog # VCTS023)
• Deionized H₂O
• 1X PBS: 0.145 M NaCl, 0.0027 M KCl, 0.0081 M Na₂HPO₄, 0.0015 M KH₂PO₄, pH 7.4

Equipment

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

Procedure

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, vegetable steamer, or pressure cooker. Note: Heating may cause cracking of glass staining dishes.
3. Immerse slides with tissues into preheated Retrieval Solution and incubate for 20 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. Cryostat sections are less resistant to the damaging effects of Retrieval Solution than paraffin-embedded tissues. To avoid tissue damage, it may be necessary to reduce the incubation time to 2-5 minutes for cryostat sections.
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 (approximately 20 minutes).
5. Gently rinse the slides with deionized water and then re-rinse 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.