Although fixation is essential for the preservation of tissue morphology, this process can also have a negative impact on IHC/ICC detection. Fixation can alter protein biochemistry such that the epitope of interest is masked and can no longer bind to the primary antibody. Masking of the epitope can be caused by cross-linking of amino acids within the epitope, cross-linking unrelated peptides at or near an epitope, altering the conformation of an epitope, or altering the electrostatic charge of the antigen. Antigen retrieval refers to any technique in which the masking of an epitope is reversed and epitope-antibody binding is restored.
Antigen Retrieval Techniques
The need for antigen retrieval depends on multiple variables, including but not limited to, the target antigen, the antibody used, the type of tissue, and the method and duration of fixation. Because they recognize multiple epitopes, polyclonal antibodies are more likely than monoclonal antibodies to detect a given antigen without the use of antigen retrieval techniques.
There are multiple techniques to restore the immunoreactivity of an epitope. Methods as simple as changing the pH or the cation concentration of the antibody diluent can influence the affinity of an antibody for its epitope. For partially masked epitopes it may be appropriate to first try increasing the primary antibody incubation conditions before commencing antigen retrieval. However, this step will also require further optimization in terms of appropriate antibody concentration, incubation time, and temperature. When discussing antigen retrieval methods, techniques generally fall into two main categories, protease-induced epitope retrieval (PIER) and heat-induced epitope retrieval (HIER). Once optimized, the effects of antigen retrieval can be pronounced.
Protease-induced Epitope Retrieval (PIER)
In the PIER method, enzymes including Proteinase K, Trypsin, and Pepsin have been used successfully to restore the binding of an antibody to its epitope. The mechanism of action is thought to be the cleavage of peptides that may be masking the epitope. The disadvantages of PIER are the low success rate for restoring immunoreactivity and the potential for destroying both tissue morphology and the antigen of interest.
Heat-induced Epitope Retrieval (HIER)
HIER is believed to reverse some cross-links and allows for restoration of secondary or tertiary structure of the epitope. The protocol must be optimized for each tissue, fixation method, and antigen to be studied. In general, HIER has a much higher success rate than PIER. HIER is performed using microwave ovens, pressure cookers, vegetable steamers, autoclaves, or water baths. Microwaves are an increasingly popular appliance for HIER. These protocols tend to involve 5 minute periods of heat followed by replacement of the buffer. For all HIER methods, slides must be cooled before commencing IHC/ICC incubations. HIER is especially time-, temperature-, buffer-, and pH-sensitive, and the best method must be determined empirically.
Protocol for Heat-induced Epitope Retrieval (HIER)
Optimization & Limitations
Antigen retrieval may require increased adherence of the sample to the slide or coverslip. In addition, the technique is often too harsh for cryostat tissue sections and alcohol-fixed tissue. The time, temperature, and pH must also be optimized for each appliance (i.e. 5-10 minutes at 92-95 °C in a water bath, versus 1-5 minutes at 120 °C in a pressure cooker). To optimize antigen retrieval preliminary studies must be conducted using a matrix of time, temperature, and pH combinations. The possibility of artifactual staining should always be considered when using any antigen retrieval methodology. The use of controls to demonstrate specific antibody binding should be included since staining is influenced by multiple variables in any given experiment.
R&D Systems offers reagents to improve tissue antigen detection and enhance immunoreactivity. During initial optimization, investigators can compare samples treated with neutral pH 7.0 Universal Antigen Retrieval Solution (Catalog # CTS015) with a slide of the same tissue without antigen retrieval. Subsequent tests using acidic or basic antigen retrieval solutions may be necessary depending on the tissue. At R&D Systems, we find that treatment with Basic Antigen Retrieval reagent (Catalog # CTS013) is frequently successful. We also offer Acidic (Catalog # CTS014) and Sampler (Catalog # CTS016) Antigen Retrieval Reagents. Acidic and basic antigen retrieval solutions are more likely to affect tissue morphology than a neutral solution.
| 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 |
This table 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.
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| Antigen Retrieval Improves Detection of p27. IHC images show the detection of p27 in paraffin-embedded human prostate cancer sections following incubation of tissue for 10 minutes at 95 °C in the specified antigen retrieval solution. Compared to no HIER treatment, p27 detection was enhanced following incubation in neutral (pH 7.0) and basic (pH 9.5, Catalog # CTS013) but not acidic (pH 5.0, Catalog # CTS014) antigen retrieval solution. P27 was detected using anti-human/mouse/rat p27 monoclonal antibody (Catalog # MAB22561; brown). |