IHC Antigen Retrieval Protocol - Creative Diagnostics

The Significance of Antigen Retrieval

Formalin-fixed tissues, commonly used in histopathology, undergo a fixation process that forms methylene bridges, cross-linking proteins, and masking antigenic sites. This cross-linking hinders the access of antibodies to their target antigens during immunohistochemical staining. Antigen retrieval methods are employed to disrupt these methylene bridges and unmask the hidden antigenic sites, enabling effective antibody binding. Commonly used methods are heat-induced epitope retrieval (HIER) and protease-induced epitope retrieval (PIER).

Fig. 1 Additional step in antigen retrieval. (Vinod K R, et al., 2016)

Protease-induced Epitope Retrieval (PIER)

As the earliest antigen retrieval method, commonly used digestive enzymes are trypsin, pepsin, and proteinase K. The mechanism of PIER is thought to be the cleavage of peptides that may mask the epitope. The action of enzymes is easily affected by temperature, which may destroy tissue morphology and target antigens, thus PIER is gradually replaced by HIER. However, PIER remains irreplaceable for individual antigens such as immunoglobulins, complements, and in formalin-fixed, paraffin-embedded renal biopsies.

Trypsin is suitable for most antigens that require retrieval after formalin/PFA fixation. The commonly used PIER methods are the pipetting method and the immersion method.

Pipetting method

Preheat trypsin to 37°C. Carefully blot the water around the tissue section, then pipette the enzyme solution and drip it onto the slice (generally 50-100 uL). Use the tip of the pipette to spread the enzyme solution over the entire section, being careful not to damage the tissue.

Place slides in a humidifier and incubate at 37°C. Avoid placing the slides directly on the shelf of the incubator, otherwise, the staining quality will be affected due to uneven temperature.

It is best to preheat the container holding the slides before putting them in the incubator.

After 10-20 minutes, take out the slide and rinse with running water for 3 minutes.

Continue with IHC staining protocol.

Immersion method

Set the temperature of the water bath to the optimum temperature for the enzyme. Add ultrapure water to the two containers holding the slide racks. Place the container in a water bath to heat.

Put the slide in one of the containers of the water bath to heat.

Prepare the enzymatic antigen retrieval buffer from warm water in another container, then return the buffer container to the water bath to reheat.

Place the heated slide in the enzyme solution for 10-20 minutes with intermittent slow shaking. Remove the slide and rinse it under running water for 3 minutes to rinse off the enzyme.

Continue with IHC staining protocol.

Heat-induced Epitope Retrieval (HIER)

HIER is a high-temperature, high-pressure method combined with a suitable antigen retrieval solution to re-expose the antigenic sites blocked by the fixative solution and increase the positive detection rate of antibodies. Sodium citrate buffer is a common choice for antigen retrieval. It effectively breaks methylene bridges and facilitates antibody binding, compatible with most antibodies. Tris-EDTA buffer and EDTA buffer are also frequently used for antigen retrieval. These buffers are relatively more repairing and suitable for most phosphotyrosine-specific antibodies.

Sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0)	1 mM EDTA, pH 8.0	Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0)
Trisodium citrate (dihydrate) 2.94 g Distilled water 1 L Mix to dissolve Adjust pH to 6.0 with 1N HCl Add 0.5 mL Tween 20 and mix well	EDTA 0.37 g Distilled water 1 L Adjust to pH 8.0 with NaOH	Tris 1.21 g EDTA 0.37 g Distilled water 1 L Mix to dissolve Adjust the pH to 9.0 Add 0.5 mL of Tween 20 and mix well

Pressure cooker method

Add the appropriate antigen retrieval buffer into the pressure cooker, then put the cooker on the heating plate and turn it on to the maximum power, do not close the lid tightly at this time. While waiting for boiling, dewax and rehydrate the sections.

After boiling, put the slides into the pressure cooker, close the lid tightly, and be careful of burns.

After the pressure cooker reaches maximum pressure, maintain it for 3 minutes.

Turn off the heating plate and remove the pressure cooker.

Release the steam and cool the cooker with cold water. After the pressure drops, open the lid and let it cool in cold water for 10 minutes. Be careful of burns.

Continue with IHC staining protocol.

Microwave method

Do not use household microwave ovens for the microwave method. There is no high-pressure environment, and the repair time is long, which will dissociate the slices. A scientific microwave is more appropriate.

Deparaffinize and rehydrate the sections.

Add the appropriate repair buffer to a microwave-safe container.

Place the slides in a container and place them in the microwave. Set the temperature at 98°C for 20 minutes to retrieve the antigen.

Take out the container and place it in water to cool for 10 minutes. Be careful of burns.

Continue with IHC staining protocol.

The IHC antigen retrieval protocol is a critical step in achieving accurate and reliable results in immunohistochemistry. By employing appropriate methods and buffer solutions, researchers can effectively unmask antigenic sites and enhance the antibody binding. Creative Diagnostics provides high-quality solutions for researchers in the life sciences industry. In addition to the antigen retrieval reagent mentioned above, we offer a comprehensive range of primary antibodies, secondary antibodies, and detection systems for IHC. These products are meticulously developed and validated to ensure excellent performance in immunohistochemical experiments.

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