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