Antigen retrieval what is

Antigen retrieval per se is the process causing protein denaturation in tissues, just like many other protein inactivation processes Table 1. Enzyme digestion may etch the masking parts of proteins around an antigen to expose its epitope. Although enzyme digestion is relatively simple and its treatment condition easy to control, the results are not necessarily dramatic and consistent depending on the types or lots of enzymes.

For example, pepsin digestion gave the best results in the immunostaining of bromo-deoxyuridine BrdU and proliferating cell nuclear antigen PCNA , whereas other enzymes had little effect Table 5.

Heating may also cleave the polypeptide backbone and disrupt the cross-links produced by fixation. The heating effect on antigen retrieval is temperature-dependent and seems to be proportional to the product of temperature and time.

In the case of PCNA immunostaining on paraformaldehyde-fixed paraffin-embedded sections, heating at 90 degrees C for at least 3 min was required, however, as the heating condition became more severe, non-specific background stains also increased Table 6 , which is one of the most serious problems in antigen retrieval Fig. In the enzymatic method, proteases such as proteinase K, trypsin, and pepsin are used.

The heat-induced method uses heat from either microwave, pressure cooker, steamer, waterbath, or autoclave and a selection of buffers. Some antigens require a combination of heating and enzyme digestion. Antigen retrieval may not be required for frozen sections where fixation is mild. The following figure illustrates the benefit of optimizing antigen retrieval to improve staining.

Effect of pH on heat-mediated antigen retrieval in human tissues From Emoto et al. J Histochem Cytochem 53 11 Buffers for heat-induced antigen retrieval. One step format antigen retrieval buffers contain detergents to allow deparaffinization and antigen retrieval to be performed in a single step to save time. IHC guide: introduction. Tips for designing a successful IHC experiment.

IHC guide: sample prep. Tissue fixation, embedding and sectioning. US: 1- customerservice biogenex. Antigen retrieval and how does it work by Admin Feb 25, ihc. What is antigen retrieval and how does it work? Figure 1. Antigen retrieval method. Antigen retrieval immunohistochemistry: review and future prospects in research and diagnosis over two decades. J Histochem Cytochem. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

Immunohistochemistry for pathologists: protocols, pitfalls, and tips. J Pathol Transl Med. A simple and effective heat induced antigen retrieval method. Garbage in, garbage out: a critical evaluation of strategies used for validation of immunohistochemical biomarkers. Mol Oncol. Analytical parameters.