Antigen/Epitope Retrieval

HIER is the most commonly used method. It involves baking or microwaving the tissue sections in the presence of an antigen retrieval solution such as citrate or EDTA buffer. When using HIER, the choice of the buffer is important. Citrate, at pH 6, tends to unmask epitopes more conservatively than EDTA at pH 9. While EDTA can result in a more complete epitope unmasking, it can also increase background signal. Use serial sections to test which solution gives the best results, with proper controls to check for nonspecific staining.

PIER is most commonly performed with proteinase K, trypsin, or pepsin. PIER is much more aggressive than HIER and can destroy delicate morphological or antigenic features. This aggressiveness makes it unsuitable for delicate samples, but perfect for over-fixed, desiccated, or stubborn samples. PIER is generally not recommended for most sample types, as the intensity of the protein degradation is usually unnecessary to effectively unmask antigens.