Antigen retrieval techniques in immunohistochemistry: comparison of different methods

Area of Science:

• Histopathology
• Immunohistochemistry
• Biomedical Research

Background:

• Immunohistochemistry (IHC) relies on antigen retrieval (AR) to unmask epitopes in fixed tissue sections.
• Formalin-fixed, paraffin-embedded (FFPE) tissues often require AR for optimal antibody binding.
• Various AR methods exist, including heat-induced epitope retrieval (HIER) and enzymatic digestion.

Purpose of the Study:

• To evaluate the efficacy of different heat-based antigen retrieval solutions and methods.
• To compare heat-based AR with enzymatic digestion for routine IHC.
• To identify optimal AR conditions for a wide range of antibodies.

Main Methods:

• Tested 61 antibodies on routine tissue sections (formalin or B5 fixed, including bone marrow biopsies).
• Applied heat-induced epitope retrieval (HIER) using citrate buffer, Tris-HCl, or EDTA solutions via microwave or pressure cooking.
• Compared results with no AR and protease XIV digestion using alkaline phosphatase/anti-alkaline phosphatase (APAAP) and streptavidin-biotin complex (SABC) methods.

Main Results:

• All but one antibody benefited from antigen retrieval.
• Heat-based AR was optimal for 55 antibodies, while protease XIV was gold standard for five.
• EDTA solutions demonstrated superior staining intensity and cell marking compared to citrate or Tris buffers.
• Pressure cooking offered practical advantages in time-saving and simultaneous slide handling.

Conclusions:

• Heat-based antigen retrieval is crucial for optimizing IHC staining with most antibodies.
• EDTA solutions are highly effective for HIER, yielding superior results.
• Pressure cooking is a convenient and efficient method for HIER in routine diagnostics.