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IHC Antigen Retrieval - What is it and when should you use it?


It goes without saying that at some point in performing immunohistochemistry (IHC) experiments on formalin-fixed paraffin embedded (FFPE) tissue, you will come across an antibody that just does not seem to work. After carefully checking to make sure you properly titered your primary antibody, confirmed your secondary antibody does recognize the species in which your primary antibody was made, and tried digesting your tissue with multiple enzymes, you might give up ever trying to get this antibody to work!

Heat-Induced Epitope Retrieval (HIER)

No need to give up yet, however, thanks to work of researchers in the early 1990’s who discovered and published the technique of heat-induced epitope retrieval (HIER), commonly called antigen retrieval. Antigen retrieval is performed by immersing your glass slide with bound tissue into a HIER buffer and subjecting it to heat in a water bath, pressure cooker, steamer, or microwave.

There are three things to remember for successful antigen retrieval:

  • Optimal heating times must be determined for each antigen.
  • The tissue on the slide must be kept hydrated.
  • The specific type of antigen retrieval solution (high or low pH) must be determined.

Using a heated buffer solution prior to incubation with your antibody of interest often unmasks the antigen of interest, allowing successful binding of your antibody to the target protein.

So why does HIER work?

During tissue processing, aldehyde-containing fixatives are used which cause cross-linking of the proteins within the tissue sample. This extensive cross-linking can mask the protein epitope that your antibody would normally recognize. HIER works by causing a reversal or unfolding of proteins within the tissue leading to unmasking of the epitope, allowing the antibody to successfully bind.

IHC Antigen Retrieval Solution – High pH

The example below shows staining on formalin-fixed paraffin embedded human pancreas sections after using IHC Antigen Retrieval Solution – High pH (10X). Left panel shows staining using 10 ug/mL of Mouse IgG1 K Isotype Control or 10 ug/mL Anti-Human CD45 Purified (right) followed by Anti-Mouse IgG Biotin, and DAB visualization. Nuclei are counterstained with hematoxylin.

ihc antigen retrieval high ph

IHC Antigen Retrieval Solution - Low pH

The example below shows staining on formalin-fixed paraffin embedded human tonsil sections after using IHC Antigen Retrieval Solution – Low pH (10X). Left panel shows staining using 5 ug/mL of Mouse IgG2a K Isotype Control eFluor® 615 or 5 ug/mL Anti-Human CD20 eFluor® 615 (right). Nuclei are counterstained with DAPI.

ihc antigen retrieval low ph

The use of HIER has made a dramatic difference in the number of antibodies that can now be used for FFPE IHC applications. 



how much necessary it is  
should it be done routinely in every IHC and how much cost of the experiment is increased 
Posted @ Sunday, November 04, 2012 12:17 PM by inderpalsingh
IHC antigen retrieval should be performed on most formalin-fixed tissue prior to immunohistochemical staining as a result of the formation of methylene bridges during fixation, which cross-link proteins thereby masking antigenic sites. Heating via microwave, pressure cooker or steamer in addition to an antigen retrieval solution is the most common method used. eBioscience offers a low pH (Cat. No. 00-4955) and high pH (Cat. No. 00-4956) antigen retrieval solution for optimum results. 
There is also an enzymactic procedure, used when heat retrieval fails, which is known to destroy epitopes and tissue morphology.  
Increased cost to the experiment is minimal. 
Posted @ Monday, November 05, 2012 3:22 PM by Wendy Parenteau
Hi, could you please send me the complimentary pocket protocol guide 
Thanks much!
Posted @ Thursday, April 04, 2013 5:56 PM by Janson Emmanual
Thank you for your request. Please complete the online application form to request the promotional code which should be used to place your order for the complimentary guide. 
Posted @ Friday, April 05, 2013 9:16 AM by Wendy Parenteau
How should I determine it's low pH or high pH antigen retrieval solution to be used? Thank you!
Posted @ Friday, January 17, 2014 9:47 AM by Rebecca
Great question! There is no definitive rule as to if a particular antigen will require additional antigen retrieval and whether that antigen retrieval would be best performed using high or low pH. Our IHC products for formalin-fixed paraffin embedded tissue are tested and validated using both high and low pH antigen retrieval methods and the technical data sheet should specify whether low or high pH antigen retrieval is required. In the event that the information is not listed, please contact technical support ( Thank you! 
- Sara, eBioscience R&D
Posted @ Friday, January 17, 2014 5:28 PM by Diane Gaige
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