Antigens can be masked as a result of the fixation process, which makes antibody binding impossible. The unmasking can be reversed with a technique called antigen retrieval/antigen unmasking, which is either mediated by heat (HIER; heat-induced antigen retrieval) or proteases (PIER; proteolytic-induced antigen retrieval). The latter uses enzymes such as proteinase K, trypsin and pepsin. The PIER method acts by degrading the peptides masking the epitope. However, PIER might also result in alterations to the specimen morphology or the antigen itself. Consequently, PIER is less frequently used than HIER, which acts by restoring the secondary and tertiary structure of an epitope.
In order to establish whether antigen retrieval should be performed and by which method the following guidelines should be followed:
Perform a literature search to determine how other researchers have visualized your antigen of interest.
Check if the antibody supplier recommends a specific antigen retrieval protocol.
If no specific protocol is available, we recommend using a HIER rather than a PIER protocol.
For HIER always start with a neutral antigen retrieval buffer, such as AbD Serotec's BUF025A and compare to a sample for which no antigen retrieval was performed.
If the neutral staining solution did not yield a good staining, alkaline or acidic antigen retrieval buffers should be tested In addition to pH, other parameters to be optimized are temperature and duration. Ideally try various pH, temperature and time combinations.
In order to exclude artifacts caused by the HIER process always include a control sample for which staining without the HIER step was performed.