Protease-induced Antigen Retrieval Protocol

  • 6 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 antigen retrieval. If at first you don't succeed, try again using the protease-induced epitope retrieval protocol below.

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.

Protease-induced Epitope Retrieval (PIER) Protocol

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

Reagents Required

Product Preparation
0.1 N Hydrochloric Acid Solution (for pH adjustment)  
0.1 N Sodium Hydroxide Solution (for pH adjustment)  
Alpha-Chymotrypsin (type II from Bovine pancreas) 0.1 g  
Calcium Chloride 0.1 g  
Methanol Industrial Methylated Spirits (IMS) or Methanol  
UltraPure Sterile Water  


  1. Set a water bath to read 37°C. Add the required amount of UltraPure Water into each trough and then place the troughs into the water bath. Allow the UltraPure Water to warm to 37° C.
  2. Dewax and rehydrate 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 for 3 minutes.
    Note: At no time from this point onwards should the slides be allowed to dry out.
  3. Place slides in one trough of UltraPure Water at 37°C to warm.
  4. Remove the other trough and into this dissolve the calcium chloride and freshly prepared chymotrypsin using a magnetic stirrer. Once dissolved, pH to 7.8 using the sodium hydroxide and hydrochloric acid solutions. Return the trough to the water bath and allow this enzyme solution to re-heat to 37°C.
  5. Transfer the warmed slides into the enzyme solution for a suggested 20 minutes then remove the slides and place them into cold running tap water for 3 minutes.
  6. Continue with the immunohistochemical staining protocol.