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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
If we using rabbit monoclonal antibody, should we still use antigen retrieval in our ihc(p) procedur?
Posted @ Saturday, September 27, 2014 1:21 AM by Nadia Purnama Dewi
Good question! The use of antigen retrieval in an IHC protocol using FFPE tissue depends on both the antigen or protein target as well as the antibody. During the fixation process the antigen can become highly cross-linked making it unrecognizable to the antibody. The antigen retrieval process restores the ability of the antibody to recognize the antigen. Rabbit and mouse monoclonal antibodies as well as polyclonal antibodies may require antigen retrieval in order to recognize and bind the protein of interest. If you have already tried staining without using antigen retrieval and do not see signal in a tissue that you know should have signal, I would try using the low and high pH antigen retrieval method. You would want to try the low pH antigen retrieval first since most antibodies that require antigen retrieval work with this protocol. Hope this helps.
- Sara, eBioscience R&D
Posted @ Tuesday, October 07, 2014 3:41 PM by Diane Gaige
for detection of methylation or hydroxymethylation on DNA should I do an antigen retrieval procedure?
Posted @ Friday, June 12, 2015 3:54 AM by F. M.
Antigen retrieval is required for the detection of methylated or hydroxymethylated DNA on formalin-fixed paraffin embedded tissues. The specific type of antigen retrieval solution, low or high pH, must be determined for the target of interest. For immunocytochemistry, fixation and permeabilization of cells is required for the detection of methylated or hydroxymethylated DNA. The type of fixative and detergent must be determined for the target of interest. For detailed immunohistochemistry and immunocytochemistry protocols, please go to the “Resources” tab on our site and click on “Best Protocols” or click on the following link: 
- Lisa, eBioscience R&D 
Posted @ Thursday, June 18, 2015 12:12 PM by Garima Mehta
my tissue is embedded in gelatin and gelatin will melt at high temp. I need to perform the antigene retriaval step at low temperture (50 deg) have you tried lower temperature? and is it effective? any thoguths on this? 
It there any other antigene retrival method besides HIER that you recommend? 
Posted @ Wednesday, July 22, 2015 11:17 AM by Seti Moughadam
An antigen retrieval step would only be required if your tissues were fixed prior to embedding in gelatin. Once your tissues are sectioned and adhered to a slide, the gelatin is usually washed away prior to beginning any kind of an antigen retrieval step, so any issue regarding temperature should not be a concern. It is important to determine whether a specific type of antigen retrieval method will be compatible with the particular target of interest and antibody being used. General heat induced epitope retrieval (HIER) protocols use temperatures at 90-100°C, but some investigators have had success with antigen retrieval protocols that use lower temperatures, 60-80°C, and longer incubation times. Other options include room temperature epitope retrieval (RTER) and proteolytic induced epitope retrieval (PIER). RTER protocols involve treatment of the tissues with acid, such as hydrochloric acid or formic acid. PIER protocols involve enzymes such as Proteinase K, Trypsin, or Pronase, and can be performed at room temperature or 37°C.  
- Lisa, eBioscience R&D 
Posted @ Monday, August 03, 2015 11:22 AM by Garima Mehta
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