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Antigen retrieval techniques in immunohistochemistry: comparison of different methods
S A Pileri1, G Roncador, C Ceccarelli
1Second Service of Pathologic Anatomy, Bologna University, Italy.
Insights
Heat-based antigen retrieval (AR) significantly improves antibody staining in tissue sections. EDTA solutions were superior to citrate or Tris buffers for AR, enhancing both staining intensity and cell marking across various immunohistochemical techniques.
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.
Abstract:
Routine sections of normal and pathological samples fixed in 10 per cent buffered formalin or B5, including EDTA-decalcified bone-marrow biopsies, were tested with 61 antibodies following heating in three different fluids: 0.01 M citrate buffer (pH 6.0), 0.1 M Tris-HCl (pH 8.0), and 1 mM EDTA-NaOH solution (pH 8.0). The sections underwent either three cycles of microwave treatment (5 min each) or pressure cooking for 1-2 min. The alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique was used as the standard detection method; with 16 antibodies a slightly modified streptavidin-biotin complex (SABC)-immunoperoxidase technique was applied in parallel. The results obtained were compared with those observed without any antigen retrieval (AR), or following section digestion with 0.05 per cent protease XIV at 37 degrees C for 5 min. Chess-board titration tests showed that all antibodies but one profited by AR. Protease XIV digestion represented the gold standard for five antibodies, while 55 produced optimal results following the application of heat-based AR. By comparison with the other fluids, EDTA appeared to be superior in terms of both staining intensity and the number of marked cells. These results were independent of tissue processing, immunohistochemical approach, and heating device. Pressure cooking was found to be more convenient on practical grounds, as it allowed the simultaneous handling of a large number of slides and a time saving of 1 min 30 s, representing the proper time for the treatment.