Skip to main content

IHC antigen retrieval protocol

Learn about the two methods of antigen retrieval: heat-mediated (also known as heat-induced epitope retrieval or HIER) and enzymatic.
Last edited Tue 31 Jan 2023

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.

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.

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

Materials and reagents

  • 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

1

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.
2

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

3

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

4

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

5

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

6

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.
7

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.

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.

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

Materials required

Pipetting method: materials and reagents

  • 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 (for trypsin, see below)

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

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

Steps

1

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.
2

Deparaffinize and rehydrate sections.

  • Place slides in one water container to warm.


3

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.
4

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.
5

Continue with immunohistochemical staining protocol.