IHC antigen retrieval protocol

Antigen retrieval is a critical step in immunohistochemistry (IHC) that restores antigenicity in formalin-fixed tissues, including fixed paraffin-embedded tissue, which is commonly used for morphological preservation. This protocol covers antigen retrieval methods such as heat-induced epitope retrieval (HIER) and enzymatic retrieval, to address different tissue types and antigens. These techniques break protein cross-links formed during fixation, allowing antibodies to bind effectively. The protocol includes steps for using pressure cookers, microwaves, and steamers, as well as enzymatic digestion using proteases. Abcam also offers pre-formulated buffers and a universal reagent kit to simplify the process. This guide ensures optimal staining results and supports reproducible IHC workflows for a wide range of tissue types and antigens.

Introduction

Formalin fixation preserves tissue morphology but leads to antigen masking, making them inaccessible to antibodies for IHC. Antigen retrieval reverses this masking by breaking methylene bridges formed during fixation. The exact mechanism of antigen retrieval is still under investigation and may involve multiple chemical processes, such as hydrolytic cleavage of formaldehyde cross-links, unfolding of epitopes, and calcium ion extraction. This protocol provides a structured approach to antigen retrieval using either heat or enzymes. The method chosen depends on the tissue type, antigen, and antibody used, and selecting the optimal method often requires empirical testing. This protocol is essential for researchers aiming to achieve high-quality, specific staining in IHC applications. It also includes buffer preparation guidelines and equipment recommendations to streamline the retrieval process.

Background and principles

Antigen retrieval is based on restoring epitope accessibility in fixed tissues. The antigen retrieval technique is a method used to unmask antigens in formalin-fixed tissues, enabling improved detection in immunohistochemistry. Formalin fixation creates cross-links that obscure antigenic sites. Heat-induced epitope retrieval (HIER) uses high temperatures and specific buffers to break these cross-links. The selection of the appropriate HIER buffer and HIER buffers, such as citrate, Tris, or EDTA, with optimal pH and composition, is crucial for maximizing antigen retrieval efficiency while minimizing tissue damage. Heat treatment can restore epitope conformation that has been altered during fixation, thereby improving antibody binding. Enzymatic retrieval employs proteases to digest masking proteins; this process, known as enzyme digestion, disrupts cross-links formed during fixation to facilitate antibody access, though it may risk tissue damage or non-specific staining. The choice of method and buffer pH is antigen-dependent and often requires empirical optimization. Abcam’s protocol emphasizes reproducibility and includes validated reagents to ensure consistent results across experiments.

Convenient buffers for confident results

For robust results with heat-mediated antigen retrieval, we recommend our pre-formulated antigen retrieval buffers. These include the three most commonly used buffers - choose from our Citrate buffer kit,  Tris-EDTA buffer kit, or  EDTA buffer kit; or  Tris buffer kit.

We also offer a  Universal Heat-mediated Antigen Retrieval Reagent kit (used with our leading PD-L1 clone 28-8) that is compatible with most antibodies and removes the need for multiple buffers.

Alternatively, you can prepare your own buffers and solutions and use the same recommended methods below.

Buffer solutions for heat-induced epitope retrieval

The following solutions are three of the more popular buffers for HIER. In the absence of datasheet information, the choice of retrieval buffer is best determined by trial.

Sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0)

• Tri-sodium 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. Store at room temperature for 3 months or at
• 4°C for longer storage.

1 mM EDTA, pH 8.0

• EDTA 0.37 g
• Distilled water 1 L
• Adjust to pH 8.0 with NaOH
• Store at room temperature for 3 months

Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0)

• Tris 1.21 g
• EDTA 0.37 g
• Distilled water 1 L
• Mix to dissolve. Adjust pH to 9.0.
• Add 0.5 mL of Tween 20 and mix well. Store at room temperature for 3 months or at 4°C for longer storage.

Stage 1 - Heat-induced epitope retrieval (HIER)

Heat-induced epitope retrieval is often performed using a pressure cooker, a microwave, or a vegetable steamer. Some labs use a water bath set to 60°C and incubate the slides in retrieval solution overnight. This is useful when working with tissue sections that fall off the slide when heated at higher temperatures, particularly bone, cartilage, and skin.

Slides should be placed in a metal rack for this procedure.

A control experiment is recommended to optimize retrieval time, where slides of the same tissue section are retrieved for 1, 2, 3, 4 and 5 min before being immunohistochemically stained.

Materials required

• Domestic stainless steel pressure cooker
• Hot plate
• Vessel with slide rack to hold approximately 400–500 mL
• Antigen retrieval buffer (Tris/EDTA pH 9.0, sodium citrate pH 6.0, or other)

Steps

Add the appropriate antigen retrieval buffer to the pressure cooker.

• Place the pressure cooker on the hotplate and turn it on full power.
• While waiting for the pressure cooker to come to a boil, de-paraffinize and rehydrate the sections.

Once boiling, transfer the slides from the tap water to the pressure cooker.

Secure the pressure cooker lid as per the manufacturer’s instructions.

As soon as the cooker has reached full pressure, time 3 min.

When 3 min have elapsed, turn off the hotplate and place the pressure cooker in an empty sink.

Activate the pressure release valve and run cold water over the cooker.

• Once de-pressurized, open the lid and run cold water into the cooker for 10 min.

Continue with the immunohistochemical staining protocol.

The use of a domestic microwave is inadvisable. Hot and cold spots are common, leading to uneven antigen retrieval. Antigen retrieval times are usually longer, due to the absence of a pressurized environment, nearly always leading to section dissociation. A scientific microwave is more appropriate. Most brands have onboard pressurized vessels and can keep the temperature at a constant 98°C to avoid section dissociation.

When using this method, it is possible for the buffer to boil over, and a large amount of the retrieval buffer will evaporate. Be sure to watch the buffer level of the slide vessel, and add more buffer if necessary. Do not allow the slides to dry out.

NOTE: Slides should be placed in a plastic rack and vessel for this procedure. Standard glass histology staining racks and vessels will crack when heated.

Materials required

• Scientific microwave or domestic (850 W)
• Microwaveable vessel with slide rack to hold approximately 400–500 mL or Coplins jar
• Antigen retrieval buffer (eg Tris/EDTA pH 9.0, sodium citrate pH 6.0, etc.)

Steps

Deparaffinize and rehydrate the sections.

Add the appropriate antigen retrieval buffer to the microwaveable vessel.

Place the slides in the microwaveable vessel.

• Place the vessel inside the microwave.
• If using a domestic microwave, set it to full power and wait until the solution comes to a boil. Boil for 20 min from this point.
• If using a scientific microwave, program so that antigens are retrieved for 20 min once the temperature has reached 98°C.

When 20 min has elapsed, remove the vessel and run cold tap water into it for 10 min.

Continue with the immunohistochemical staining protocol.

Many labs use a vegetable steamer or rice cooker for heat-mediated antigen retrieval. The procedure is similar to microwaving in that it maintains the temperature of the buffer at 100°C, but without the vigorous boiling of the microwave method. This method may be adapted to a water bath set at 100°C in place of the steamer.

Slides should be placed in a plastic or metal rack and vessel for this procedure. Standard glass histology staining racks and vessels will crack when heated.

Materials required

• Vegetable steamer
• Vessel with slide rack to hold approximately 400–500 mL (or 250 mL if using Tissue-Tek containers)
• Antigen retrieval buffer (eg Tris/EDTA pH 9.0, sodium citrate pH 6.0)

Steps

Deparaffinize and rehydrate the sections.

Set up the vegetable steamer according to the manufacturer’s instructions and preheat.

Pre-heat the appropriate antigen retrieval buffer to boiling in a flask.

Put the container that will hold the rack of slides into the vegetable steamer.

Carefully add the hot buffer to the container, followed by the rack of slides.

• If more convenient, add the buffer to the container before placing the container in the steamer.

Close the lid of the steamer.

• The container of buffer should also have a closed lid.

Keep the container in the steamer for 20 min from this point.

When 20 min has elapsed, remove the vessel and run cold tap water into it for 10 min.

Continue with the immunohistochemical staining protocol.

Stage 2 - Enzymatic antigen retrieval

Enzymatic retrieval can sometimes damage the morphology of the section, so the concentration and treatment time need to be tested. There are at least two methods for applying the enzyme solution to the tissue: directly pipetting the solution onto the tissue on the slide, or placing a rack of tissue slides into a container of enzyme solution. The first method uses less reagent, but since each slide needs to be handled individually, the incubation time must be monitored carefully to ensure all slides receive the same treatment. For this reason, it is easier to treat large batches of slides by immersing them in a container of enzyme solution. If using an automated staining system (eg Ventana), consult the manufacturer for an appropriate enzymatic retrieval protocol.

The enzyme to use will be indicated on the antibody datasheet. If not, trypsin is useful for a wide range of antigens that require retrieval post-formalin/PFA fixation.

Be sure to read the manufacturer’s datasheet for the enzyme you choose, as some enzymes require specific buffers and cofactors for activity.

Materials required

• 37°C water bath
• Slide racks and slide rack containers
• Enzymatic antigen retrieval solution (for trypsin, see enzymatic retrieval pipetting method)

Steps

Set water bath to the optimal temperature for the enzyme you are using.

• Add ultrapure water to two containers that can hold slide racks
• Place the containers into the water bath to warm.

Deparaffinize and rehydrate sections.

• Place slides in one water container to warm.

Prepare the enzymatic antigen retrieval buffer from the warm water in the other container.

• Then, return the container to the water bath to allow the solution to re-heat.

Transfer the warmed slides into the enzyme solution for 10–20 min with intermittent gentle agitation.

• After this time, remove the slides and place them in running tap water for 3 min to rinse off the enzyme.

Continue with immunohistochemical staining protocol.

Materials required

• 37°C incubator

• Humidified chamber (either the incubator itself or a container with a wet paper towel)

• Two slide rack containers of TBS with slide rack

• Enzymatic antigen retrieval solution, choose from the relevant solution recipes below:

  • Trypsin stock solution (0.5% in distilled water)

    • Trypsin 50 mg
    • Distilled water 10 mL
    • Mix to dissolve, store at -20ºC

  • Calcium chloride stock solution (1%)

    • Calcium chloride 0.1 g
    • Distilled water 10 mL
    • Mix well and store at 4ºC

  • Trypsin working solution (0.05%)

    • Trypsin stock solution (0.5%) 1 mL
    • Calcium chloride stock solution 1% 1 mL
    • Distilled Water 8 mL
    • Adjust pH to 7.8 with 1N NaOH. store at 4ºC for one month or -20ºC for long term storage

For enzymatic antigen retrieval, we recommend our Trypsin solution kit. We also offer a Pepsin solution kit and Proteinase K solution kit.

Steps

Prepare the trypsin solution and pre-heat to 37°C.

• Carefully blot excess water from around the tissue section and pipette the enzyme solution (generally 50–100 μL will suffice) onto the section.

Place the slides in a humidified container and then into the 37°C incubator.

After 10–20 min, remove the slides from the incubator and transfer to a rack in a container of tap water.

• Rinse by running tap water for 3 min.

Continue with immunohistochemical staining protocol.

Comparison of methods

HIER and enzymatic antigen retrieval differ in mechanism and application, especially when working with paraffin-embedded tissue sections. HIER is gentler and more consistent, using heat and buffers like citrate or Tris-EDTA. PIER employs an enzyme solution containing enzymes such as trypsin or proteinase K to digest tissue and unmask antigens, which can be more aggressive and risk tissue damage. HIER is preferred for most antigens due to its controlled conditions, while enzymatic antigen retrieval is useful for difficult-to-retrieve epitopes in paraffin-embedded tissue sections. We recommend testing both methods to determine the optimal approach for each antigen, ensuring reliable and specific staining.

Optimization of antigen retrieval

Optimizing the antigen retrieval step is essential for achieving consistent and high-quality immunohistochemical staining. The optimal antigen retrieval method depends on several factors, including the type of tissue, the nature of the antigen, and the primary antibody used. Variables such as the duration and type of formalin fixation, the age of the tissue sample, and the choice of buffer solution can all impact the effectiveness of epitope retrieval. For heat-induced epitope retrieval, parameters like temperature, heating duration, and the cooling process must be carefully adjusted to maximize antigen exposure while preserving tissue morphology. Standardizing the retrieval method across experiments helps reduce variability in staining intensity and background staining, which is particularly important in diagnostic pathology and research settings. By systematically optimizing the antigen retrieval protocol, laboratories can enhance the sensitivity and specificity of their immunohistochemical staining, ensuring reliable and reproducible results for a wide range of tissue types and antigens.

Applications

Antigen retrieval is essential for IHC in formalin-fixed paraffin-embedded (FFPE) tissues. It enables the detection of intracellular and membrane-bound proteins by restoring antibody access. Antigen retrieval techniques are also applicable to archival tissues and can be adapted for frozen tissue sections and frozen tissue to preserve antigenicity, offering alternatives to FFPE samples. Applications include cancer biomarker detection, neuroscience, pathology, and developmental biology. This protocol supports manual and automated staining workflows and is compatible with a wide range of antibodies. The use of validated buffers and reagents ensures high sensitivity and specificity in diverse research contexts.

Limitations

While antigen retrieval enhances staining, it has limitations. Over-retrieval can damage tissue morphology or cause section loss, especially with high temperatures or prolonged enzymatic digestion. Buffer pH and retrieval time must be optimized for each antigen. Microwave-based HIER may result in uneven heating, leading to inconsistent staining. Enzymatic methods can be harsh and require precise timing. Abcam advises performing control experiments to fine-tune conditions and minimize variability.

Troubleshooting

Common issues in antigen retrieval include weak or absent staining, high background, and tissue detachment. These may result from incorrect buffer pH, insufficient retrieval time, or overheating. We recommend using validated buffers, monitoring temperature closely, and avoiding slide drying. For enzymatic retrieval, ensure enzyme activity and avoid over-digestion. Troubleshooting tips include adjusting buffer composition, using a scientific microwave for consistent and controlled heating during antigen retrieval, and performing time-course experiments to optimize conditions.