D. Antigen retrieval (IHC-P guide)

A guideline procedure and tips for staining of paraffin embedded sections, including antigen retrieval, chromogenic detection and fluorescent detection

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Most formalin-fixed tissue requires an antigen retrieval step before immunohistochemical staining can proceed. This is due to the formation of methylene bridges during fixation, which cross-link proteins and therefore mask antigenic sites. The two methods of antigen retrieval are heat-mediated (also know as heat-induced epitope retrieval, or HIER) and enzymatic.

Both antigen retrieval methods serve to break the methylene bridges and expose the antigenic sites in order to allow the antibodies to bind. Some antigens prefer enzymatic to heat mediated antigen retrieval and vice versa. Enzymatic retrieval can sometimes damage the morphology of the section, so the concentration and treatment time need to be tested. Antigen retrieval with Tris/EDTA pH 9.0 buffer is suitable for most antigens. Sodium citrate pH 6.0 is also widely used. Read this explanation of why Abcam recommends using Tris/EDTA pH 9.0 buffer before sodium citrate pH 6.0.

Heat-induced epitope retrieval is most often performed using a pressure cooker, a microwave, or a vegetable steamer. Additionally, some labs will use a water bath set to 60°C and incubate the slides in retrieval solution overnight. Unless the antigen retrieval method is stated on the antibody datasheet, the optimal method for each antigen must be found experimentally. Abcam recommends testing several methods to find the retrieval that gives optimal staining.

1. Buffer solutions for heat-induced epitope retrieval

The following solutions are three of the more popular buffers for HIER. In the absence of advice from other researchers for a particular antibody, choice of retrieval buffer is best accomplished by experiment.

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

Tri-sodium citrate (dihydrate) 2.94 g
Distilled water 1000 ml
Mix to dissolve. Adjust pH to 6.0 with 1N HCl.
Add 0.5 ml of Tween 20 and mix well. Store at room temperature for 3 months or at 4°C for longer storage.

1 mM EDTA, adjusted to pH 8.0

EDTA 0.37 g
Distilled water 1000 ml
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 1000 ml (100 ml to make 10x, 50 ml to make 20x)
Mix to dissolve. pH is usually at 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.

2. Heat-induced epitope retrieval methods

Pressure cooker

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

Materials and reagents

Domestic stainless steel pressure cooker
Hot plate
Vessel with slide rack to hold approximately 400-500 ml
Antigen retrieval buffer (i.e. Tris/EDTA pH 9.0, sodium citrate pH 6.0)

Method

  1. Add the appropriate antigen retrieval buffer to the pressure cooker. Place the pressure cooker on the hotplate and turn it on full power. Do not secure the lid of the pressure cooker at this point, simply rest it on top.
    While waiting for the pressure cooker to come to a boil, deparaffinize and rehydrate the sections as above.
  2. Once boiling, transfer the slides from the tap water to the pressure cooker. Use care with hot solution - use forceps! Secure the pressure cooker lid as in the manufacturer’s instructions.
  3. As soon as the cooker has reached full pressure (see the manufacturers instructions), time 3 min (See note i).
  4. When 3 min has elapsed, turn off the hotplate and place the pressure cooker in an empty sink.
  5. Activate the pressure release valve (see the manufacturer’s instructions) and run cold water over the cooker. Once de-pressurized, open the lid and run cold water into the cooker for 10 min. Use care with hot solution! (See note ii).
  6. Continue with the immunohistochemical staining protocol.

Notes

i. Three minutes is only suggested as a starting point antigen retrieval time. Less than 3 minutes may leave the antigens under-retrieved, leading to weak staining. More than 3 minutes may leave them overretrieved, leading to non-specific background staining and also increasing the chances of sections dissociating from the slides. A control experiment is recommended beforehand, where slides of the same tissue section are retrieved for 1, 2, 3, 4 and 5 minutes before being immunohistochemically stained to evaluate optimum antigen retrieval time for the particular antibody being used.
ii. This is to allow the slides to cool enough so they may be handled, and to allow the antigenic site to re-form after being exposed to such high temperature.

Microwave

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 much more appropriate. Most brands have onboard pressurized vessels and can keep the temperature at a constant 98°C to avoid section dissociation. The only drawback is the expense of purchasing one!

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.

Top tip Molly

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

Domestic (850 W) or scientific microwave
Microwaveable vessel with slide rack to hold approximately 400-500 ml or Coplin jar
Antigen retrieval buffer (e.g. Tris/EDTA pH 9.0, sodium citrate pH 6.0, etc.)

Method

  1. Deparaffinize and rehydrate the sections as above.
  2. Add the appropriate antigen retrieval buffer to the microwaveable vessel (See note i).
  3. Remove the slides from the tap water and place them in the microwaveable vessel. Place the vessel inside the microwave. If using a domestic microwave, set to full power and wait until the solution comes to the boil. Boil for 20 minutes from this point. If using a scientific microwave, program so that antigens are retrieved for 20 minutes once the temperature has reached 98°C. (See note ii).
  4. When 20 minutes has elapsed, remove the vessel and run cold tap water into it for 10 minutes. Use care with hot solution. (See note iii).
  5. Continue with the immunohistochemical staining protocol.

Notes

i. Use a sufficient volume of antigen retrieval solution in order to cover the slides by at least a few centimeters if using a non-sealed vessel to allow for evaporation during the boil. Be sure to watch for evaporation and for boiling over during the procedure, and do not allow the slides to dry out!
ii. 20 minutes is only a suggested antigen retrieval time. Less than 20 minutes may leave the antigens underretrieved, leading to weak staining. More than 20 minutes may leave them over-retrieved, leading to nonspecific background staining and also increasing the chances of sections dissociating from the slides. A control experiment is recommended beforehand, where slides of the same tissue section are retrieved for 5, 10, 15, 20, 25 and 30 minutes before being immunohistochemically stained to evaluate optimum antigen retrieval time for the particular antibody being used.
iii. This allows the slides to cool enough so they may be handled, and allows the antigenic site to re-form after being exposed to high temperature.

Vegetable steamer

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.

Top tip Molly

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

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

Method

  1. Deparaffinize and rehydrate the sections as above.
  2. Set up the vegetable steamer according to the manufacturer’s instructions and preheat.
  3. Pre-heat the appropriate antigen retrieval buffer to boiling in a flask (a microwave is handy for this).
  4. Put the container that will hold the rack of slides into the vegetable steamer.
  5. 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.
  6. Close the lid of the steamer. The container of buffer should also have a lid. The rack of slides will initially bring the temperature of the AR solution down but it will return to 95 - 100°C within several minutes.
  7. Keep the container in the steamer for 20 minutes from this point. (See note ii for the microwave method).
  8. When 20 minutes has elapsed, remove the vessel and run cold tap water into it for 10 minutes. Use care with hot solution. (See note iii for the microwave method).
  9. Continue with the immunohistochemical staining protocol.

3. Enzymatic antigen retrieval

Choice of enzyme will be indicated on the datasheet for the antibody. If not, trypsin has been shown to be 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 (e.g. > 5) by immersing them in a container of enzyme solution. If using an automated staining system (e.g. Ventana), consult the manufacturer for an appropriate enzymatic retrieval protocol.

Enzymatic retrieval, 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 (See section G for TBS recipe.)
Enzymatic antigen retrieval solution (For trypsin, see below. For pepsin and proteinase K, see section G)

The following method uses trypsin. There are commercially available trypsin preparations optimized for IHC, such as Abcam's Trypsin enzymatic antigen retrieval solution (ab970), or it can be prepared as follows:

Trypsin Stock Solution (0.5% in distilled water)

Trypsin 50 mg
Distilled water 10 ml
Mix to dissolve. Store at -20°C.

Calcium Chloride Stock Solution (1%)

Calcium chloride 0.1 g
Distilled water 10 ml
Mix well and store at 4@°C.

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 4°C for one month or -20°C for long term storage.

Method

  1. Prepare the trypsin and pre-heat to 37°C. Carefully blot excess water from the around the tissue section and pipet the enzyme solution (generally 50 - 100 ul will suffice) onto the section. It may be necessary to spread the solution around the section with the pipet tip; be careful not to damage the tissue.
  2. Place the slides in a humidified container and then into the 37°C incubator. Avoid placing the slides directly on the incubator shelves as there will be variations in temperature that could affect staining intensity. Ideally, the container holding the slides is pre-heated in the incubator.
  3. After 10- 20 minutes (this will need to be optimized), remove the slides from the incubator and transfer to a rack in a container of tap water. Rinse by running tap water for 3 minutes.
  4. Continue with immunohistochemical staining protocol.

Enzymatic retrieval, immersion method

Materials and reagents

37°C waterbath
Slide racks and slide rack containers
Enzymatic antigen retrieval solution (For trypsin, see pipetting method. For pepsin and proteinase K, see section G)

Method

  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. (See note ii).
  2. Deparaffinize and rehydrate sections as above. Place slides in one water container to warm (See note iii).
  3. Prepare the enzymatic antigen retrieval buffer from the warm water in the other container, and then return the container to the water bath to allow the solution to re-heat (See note iv).
  4. Transfer the warmed slides into the enzyme solution for 10 - 20 minutes (see note v) with intermittent gentle agitation, then remove the slides and place them in running tap water for 3 minutes to rinse off the enzyme.
  5. Continue with immunohistochemical staining protocol.

Notes

i. Be sure to read the manufacturer’s literature for the enzyme you choose, as some enzymes require specific buffers and cofactors for activity.
ii. Use a sufficient volume of water or buffer to cover the slides.
iii. Placing cold slides into the enzyme solution will lower the temperature of the solution, reducing enzyme activity and leading to under-retrieval of the antigenic site.
iv. Prepare the enzymatic antigen retrieval solution as quickly as possible to avoid impairing the activity of the enzyme. Allow this solution to return to temperature before introducing the slides.
v. Ten to twenty minutes is only suggested as a starting point incubation time. Less than 10 minutes may leave the antigens under retrieved, leading to weak staining. More than 20 minutes may leave them over retrieved, leading to non-specific background staining and also increasing the chances of sections dissociating from the slides or damage to the morphology of the tissue. A control experiment is recommended beforehand, where slides of the same tissue section are incubated in the enzyme solution for 10, 15, 20, 25, and 30 minutes before being immunohistochemically stained to evaluate optimum antigen retrieval time for the particular antibody being used.

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