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A Brief Guide on Antigen Retrieval Methods

Posted by Immuno Staining on November 12th, 2018

The fact is that formalin-fixed tissue needs an antigen retrieval procedure before immunohistochemical staining can begin. This is because of the formation of methylene bridges that occurs during fixation. It takeovers link proteins and thus mask antigenic sites. There are two methods recommended for Antigen Retrieval - enzymatic or heat mediated. Both of these Antigen Retrieval Methods will assist to break the methylene bridges in order to expose the sites to endow the antibodies to bind.

Moreover, the enzymatic method is considered as a gentler process than heat mediated method. Thus, it is well recommended for sensitive tissues. It should be noted that the enzymatic method takes much time than the heat mediated method. But this method is quite technically demanding. Moreover, in a case, the antigen retrieval step is stated on the antibody datasheet, start someone by trying the heat mediated method. In a case, you will not succeed, then again try with the enzymatic method.

Let’s comprehensively discuss both processes of Antigen Retrieval Method

  1. Enzymatic Antigen Retrieval Method:

In this, tissues procedure is done in the best way on amino-propyl-tri-ethoxy-silane coated slides. Moreover, these should be placed in a standard rack for this process.

Requirement of Reagents

  • Xylene
  • UltraPure Sterile Water use ROCKLAND code # MB-009-1000
  • Alpha-Chymotrypsin (type II from Bovine pancreas) 0.1 g
  • Calcium Chloride 0.1 g
  • 0.1 N Hydrochloric Acid solution (for pH adjustment)
  • 0.1 N Sodium Hydroxide solution (for pH adjustment
  • Methanol Industrial methylated spirits or (IMS)


  • Arrange a water bath and add the required amount of UltraPure Water then endow it to warm to 37 degrees C.
  • After this, make sure to Dewax and re-hydrate paraffin sections with the help of xylene or methanol.
  • Once dissolved pH to 7.8, return the trough to the water bath and endow this enzyme solution to re-heat to 37degrees C.
  • Now transfer slides into the enzyme solution before removing the slides and placing them into cold water.
  • Continue with ICC Protocol Immunohistochemical Staining.

  1. Microwave Antigen Retrieval Method

For this Antigen Retrieval Method, there will be no use of the domestic microwave. Only a scientific microwave is recommended for this.

Requirement of Reagents

  • Xylene
  • 0.1 N Sodium Hydroxide solution (for pH adjustment)
  • 0.1 N Hydrochloric Acid solution (for pH adjustment)
  • 0.2M Hydrochloric acid solution 22.0 ml
  • Tri-Sodium Citrate 2.94 g
  • UltraPure Sterile Water use ROCKLAND code # MB-009-1000
  • Methanol Industrial methylated spirits or IMS


  • First of all, Dewax and re-hydrate the paraffin sections with the help of xylene or methanol. Keep it clean until the microwave antigen retrieval solution has been prepared.
  • Then add the hydrochloric acid, trisodium citrate, and UltraPure Water together in a conical flask. Make sure to use a magnetic stirrer to verify that all reagents are completely dissolved.
  • Eliminate the slides with the help of water before placing them in the microwaveable vessel. For scientific microwave, only 15 minutes are recommended. Once done, remove the vessel and again place it under the water for 10 minutes.
  • When 15 minutes has elapsed, remove the vessel and run cold tap water into it for 10 minutes.
  • Proceed further with immunohistochemical staining protocol.

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Also See: Antigen Retrieval, Retrieval Methods, Retrieval Method, Ph Adjustment, Water, Retrieval, Method

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