Tips for Step 4 – Perform Antigen/Epitope Retrieval

Tissue fixation methods often result in antigen masking, which subsequently impairs antibody binding and therefore protein detection. The effects of tissue fixation can be partially reversed by performing epitope retrieval, also known as antigen unmasking.

Two types of epitope retrieval have been established; heat based (HIER; Heat-Induced Epitope Retrieval) or enzyme based (PIER; Proteolytic-Induced Epitope Retrieval).

Table 1: Comparison of epitope retrieval methods.




Method Overview

Can be performed using autoclaves, heating plates, hot water baths, pressure cookers, microwaves or steamers (Kim et al. 2016, Ward and Rehg 2014)

Can be performed using enzymes such as pronase, proteinase K, trypsin or pepsin (see IHC protocol 7)

Mode of Action

Restores secondary and tertiary epitope structures

Degrades the peptides masking the epitope


Very popular

Less popular compared to HIER
Reason: may induce changes to the specimen's morphology

Consider these guidelines to determine if and how to perform antigen retrieval:  

  • Perform a literature search to determine what retrieval techniques and conditions other researchers have used to successfully detect your protein of interest. This search also allows you to familiarize yourself with the expected localization/distribution pattern

  • Check if the antibody supplier or IHC resources recommend a specific antigen retrieval protocol (for example we have a dedicated protocol for the antigen retrieval of the murine F4/80 antigen; see IHC protocol 9)

  • If no specific antigen retrieval method is recommended for your specific protein, try HIER first (see Table 1)

  • For HIER, initially start with a neutral pH antigen retrieval buffer, such as Antigen Retrieval Buffer, pH 8.0 (BUF025A). Compare against tissue for which the antigen retrieval step has been omitted

  • Alternatively, if no specific protocol is recommended, start with commonly used antigen retrieval buffers such as 10 mM citrate buffer (pH 6.0) and Tris-EDTA buffer (pH 9.0) (see IHC protocol 7). You may also want to compare different buffers to determine the optimal conditions

  • In addition to pH, consider optimizing the temperature and duration of the HIER procedure. Ideally test various pH, temperature and time parameters 

  • To eliminate staining artifacts created by the HIER process, compare against a control sample for which no HIER treatment was performed 

  • Bespoke treatment conditions may be required for the detection of certain antigens such as 5′-bromo-2′-deoxyuridine (BrdU) incorporated into DNA. For BrdU staining experiments, treatment with hydrochloric acid or nucleases is frequently performed to facilitate anti-BrdU antibody binding (Liboska et al. 2012)