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Learn about the two methods of antigen retrieval: heat-mediated (also known as heat-induced epitope retrieval or HIER) and enzymatic.
Most formalin-fixed tissues require an antigen retrieval step before immunohistochemical staining. Methylene bridges formed during fixation cross-link proteins and mask antigenic sites. Antigen retrieval methods break these methylene bridges and expose antigenic sites, allowing antibodies to bind. The two methods for antigen retrieval are heat induced epitope retrieval (HIER) and enzymatic retrieval.
Enzymatic retrieval can sometimes damage the morphology of the section, so the concentration and treatment time need to be tested.
Heat-induced epitope retrieval is most 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; in particular bone, cartilage, and skin.
Unless the antigen retrieval method is stated on the antibody datasheet, the optimal method for each antigen must be found experimentally. This applies also to the choice of buffer used for heat-mediated retrieval. We recommend testing several methods to find the retrieval method that gives optimal staining.
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 use our 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.
The following solutions are three of the more popular buffers for HIER. In the absence of datasheet information, choice of retrieval buffer is best accomplished by trial.
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)
Slides should be placed in a metal rack for this procedure.
Materials and reagents
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.
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.
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 and reagents
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 and reagents
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.
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 needs to be monitored carefully for each slide to ensure all slides are receiving 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.
Pipetting method: materials and reagents
Trypsin stock solution (0.5% in distilled water)
Calcium chloride stock solution (1%)
Trypsin working solution (0.05%)
Pipetting method: method
Immersion method: materials and reagents
Immersion method: method
Be sure to read the manufacturer’s literature for the enzyme you choose, as some enzymes require specific buffers and cofactors for activity.
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