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Antigen retrieval is a technique required in most formalin-fixed tissues before immunohistochemical staining. Antigen retrieval is used to reverse epitope masking and restore epitope-antibody binding often lost during the fixation process. Avoiding this step may result in weak or false negative staining.
Some fixatives can mask epitopes and reduce antigen-antibody binding. An antigen retrieval step can restore tissue antigenicity. Antigen retrieval is usually carried out before staining to unmask hidden or denatured target epitopes. Antigen retrieval can be heat-induced or enzymatic, with factors such as temperature, pH and time. Which method to use depends on the tissue type and the primary antibody.
There are two methods of antigen retrieval: heat-induced epitope retrieval (HIER) and proteolytic-induced epitope retrieval (PIER). Which method to use depends on the tissue type and the primary antibody.
Heat-induced epitope antigen retrieval is carried out when the slide is heated up for a specific time in a specific buffer. Different sources such as a microwave, pressure cooker, water bath, steamer, etc are normally used for this step. The most commonly used buffer is the citrate buffer (see recipe in general immunohistochemistry protocols). However, the choice of buffer depends on the antibody in use. It's important to follow the instructions on the specific datasheet and recommendations for retrieval buffers. As a rule of thumb, an EDTA buffer is favorable when working with antibodies against phospho-tyrosines.
In the proteolytic-induced epitope retrieval (PIER) technique, epitopes are unmasked by peptidases.
Trypsin is a popular enzyme that breaks the protein cross-links, unmasks the hidden antigen, and thus increases the staining intensity and specificity of the primary antibody.
Prepare the trypsin and pre-heat to 37ºC. Pipette the enzyme solution onto the section.
Place the slides in a humidified container and then into the 37ºC incubator.
After 15 minutes, remove the slides from the incubator and transfer to a rack in a container with tap water. Rinse by running water for 3 minutes.
Continue with the immunohistochemical staining. (Other enzymes used are proteinase k, pepsin, protease, or pronase.)
Comparison of heat-induced epitope retrieval (HIER) and proteolytic-induced epitope retrieval (PIER)
|Advantage||Smooth epitope recovery||Preferred for difficult-torecover epitopes|
|Disadvantage||No impact on cell morphology||Impacts and damages, also a harsh method|
|Difficulties||Unequal retrieval due to unequal heating||Concentration calibration|
|pH*||Typically pH 6 (citrate), pH 9 (Tris-EDTA)||Typically pH 7.4|
|Incubation time*||Around 20 mins||Around 10 mins|
|Temperature*||Around 100ºC||Around 37ºC|
|*Optimal conditions always have to be determined by each laboratory and in accordance with the specific product information.|