The accurate and repeatable detection of a specific protein is central to successful IHC staining. Primary antibodies bind to a small region or multiple regions (epitope/s) of these very complex proteins.
During formalin fixation the protein structure is modified by formalin cross-linkages that can mask the target epitope. Before staining formalin-fixed, paraffin-embedded (FFPE) tissue, the target site may need to be “unmasked” using a process known as epitope retrieval. There are two common methods used: Heat Induced Epitope Retrieval (HIER) and Enzyme Induced Epitope Retrieval (EIER). The choice of method depends on the primary antibody used and the retrieval method suggested by the antibody manufacturer.
Both HIER and EIER follow tissue deparaffinization and rehydration. Some automated stainers include epitope retrieval steps (both HIER and EIER). These stainers reduce the overall laboratory workload as well as providing excellent results due to their consistency and temperature control. The HIER method involves incubating the sample in a buffered solution at an elevated temperature. Successful HIER depends on buffer composition, buffer pH, heating time and temperature. The most common buffers are alkaline EDTA buffer (pH 9.0) and a mildly acidic citrate buffer (pH 6.0). There are a number of different heating methods with temperatures of 100 ºC and above providing the best results. However excessive temperature can adversely affect the epitope and tissue morphology (structure).
EIER is typically conducted at 37 ºC – 40 °C using proteolytic enzymes that digest, or physically breakdown, the cross-linking bonds. The process is faster than HIER, taking around 5 – 10 minutes. The enzyme can begin to digest and destroy the samples if incubated for too long.
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