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Antigen retrieval (AR) has been a revolutionary technique in IHC and has been instrumental in unmasking low-level or formalin cross-linked antibodies. Get more information on epitope retrieval optimization tips in
the link below.
Antigen retrieval (AR) is the process of breaking protein cross-links that mask antigens in formalin-fixed tissue sections. Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, giving weak or false negative staining.
Antigen retrieval breaks the protein cross-links, enhancing staining intensity by unmasking the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections.
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The process of fixing a tissue sample with paraformaldehyde causes the formation of peptide crosslinks that serve to preserve tissue architecture and protein localization. These peptide crosslinks can also mask epitopes, making them inaccessible to antibody binding. In order to get good-quality IHC stains, these cross-links must be broken in the process known as epitope retrieval.
There are two common methods of epitope retrieval: heat-induced epitope retrieval (HIER), and proteolytic-induced epitope retrieval (PIER). Selecting the right method is critical to get the best results for your application
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.
Problems with the antigen retrieval step of IHC can result in a weak signal. Troubleshoot your IHC experiment with these tips:
You can find more IHC technical resources at Boster Bio. Download our IHC eBook.
IHC optimization is a critical step in any test. This guide gives you insight on antigen retrieval, fixation and embedding. Learn how to optimize your immunohistochemistry test to get valuable results.
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See MoreIHC images show the detection of CD3E in paraffin-embedded human tonsil sections following incubation of tissue for 5 and 15 minutes at 95°C in the specified antigen retrieval solution. If you need an acidic antigen retrieval buffer, check out Boste...
See MoreTroubleshooting guides Optimizing your Antigen Retrieval Method Greetings Earthling, Here are some technical tips for optimizing your antigen retrieval method! To find the optimal antigen recovery method, we suggest that you test both HIER and PIER m...
See MoreIHC Enzyme Antigen Retrieval Reagent is an IHC enzymatic antigen unmasking reagent composed of multiple proteinases used for enzymatic digestion of tissue and proteins in proteolytic induced epitope retrieval (PIER). AR0022
See MoreAfter saturation, the inactivated antibody replaces the primary antibody in the IHC protocol for the control. The samples are incubated with only the antibody diluent without adding the primary antibody. No Primary Controls No primary controls (aka s...
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