Heat-induced Antigen Retrieval Protocol

  • 5 steps
  • 1 hours
  • 7 reagents required

Which Antigen Retrieval Method Should I Use?

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 either protease-induced epitope retrieval (PIER) or heat-induced epitope retrieval (HIER). Both serve to break the methylene bridges and expose the antigenic sites in order to allow the antibodies to bind. Some antigens prefer the protease method to heat-induced epitope retrieval and vice versa. The protease method tends to be a much gentler process than heat-induced, so is best suited to more sensitive tissues. However, the protease method tends to take much longer and is more technically demanding.

If no antigen retrieval step is stated on the antibody data sheet, start off by trying the heat-induced epitope retrieval. If at first you don't succeed, try again using the protease-induced antigen retrieval protocol.

Frozen tissue sections do not need an epitope retrieval step. Once mounted on APES coated slides, they are best kept at -80°C until needed. When required, allow the slides to warm at room temperature for 5 minutes, then acetone fix for 5 minutes followed by a PBS or TBS rinse. Afterwards, continue with the immunohistochemical staining protocol.

Heat-induced Epitope Retrieval (HIER) Protocol

The use of a domestic microwave is inadvisable. Hot and cold spots are common which lead to uneven antigen retrieval. In addition, antigen retrieval times are usually longer, due to the absence of a pressurized environment that nearly always leads to section dissociation.

A scientific microwave is much more appropriate. Most brands have on-board 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! But if your department has one by all means use it.

Tissue sections are best mounted on APES (amino-propyl-tri-ethoxy-silane) coated slides. Slides should be placed in a standard plastic rack for this procedure.

Reagents Required

Product Preparation
0.1 N Sodium Hydroxide Solution (for pH adjustment)  
0.1 N Hydrochloric Acid Solution (for pH adjustment)  
0.2 M Hydrochloric Acid Solution 22 mL  
Methanol Industrial Methylated Spirits (IMS) or Methanol  
Tri-Sodium Citrate 2.94 g  
UltraPure Sterile Water  


  1. De-wax and rehydrate the paraffin sections by placing them in 3 changes of xylene for 3 minutes each, followed by 3 changes of IMS or methanol for 3 minutes each, followed by cold running tap water. Keep them in the tap water until the microwave antigen retrieval solution has been prepared. At no time from this point onward should the slides be allowed to dry out!
  2. Add the tri-sodium citrate, hydrochloric acid, and UltraPure Water together in a 1L beaker/conical flask. Use a magnetic stirrer to ensure that all reagents are properly dissolved. Adjust to pH 6.0 using the sodium hydroxide and hydrochloric acid solutions. Add this solution to the microwaveable vessel.
  3. Remove the slides from the tap water and place them in the microwaveable vessel. Place the vessel inside the microwave. If domestic, set to full power and wait until the solution comes to the boil. Boil for 15 minutes from this point. If scientific, program so that antigens are retrieved for 15 minutes once the temperature has reached 98°C.
  4. When 15 minutes has elapsed, remove the vessel and run cold tap water into it for 10 minutes.
  5. Continue with the immunohistochemical staining protocol.