Although fixation is essential for the preservation of tissue morphology, this process can also have a negative impact on IHC/ICC detection. Fixation can alter protein biochemistry such that the epitope of interest is masked and can no longer bind to the primary antibody. Masking of the epitope can be caused by cross-linking of amino acids within the epitope, cross-linking unrelated peptides at or near an epitope, altering the conformation of an epitope, or altering the electrostatic charge of the antigen. Antigen Retrieval Methods refers to any technique in which the masking of an epitope is reversed and epitope-antibody binding is restored.

Immunostaining is the website that offers you detailed information about IHC Protocol. It is a small part of Boster bio that gives you complete knowledge about IHC Principle and IHC Protocol. This website is especially designed to educate the people about the detailed process of immunostaining and how it works?

According to the experts at immunostaining.info formaldehyde fixation usually generates methylene bridges which cross-link proteins and therefore mask the epitope of interest. It is essential to unmask the antigen epitopes in order to allow the antibodies to bind, either by heat (Heat Induced Epitope Retrieval: HIER) or enzymatic digestion (Proteolytic Induced Epitope Retrieval: PIER).

The HIER method can be implemented by microwave, high pressure or water bath. It breaks the methylene bridges and exposes the epitope to allow the antibodies to bind by continuously heating. The following antigen retrieval reagent is required:

You can find IHC Antigen Retrieval Optimization Tips at the resource immunostaining.info. While selecting the right antigen retrieval method, 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.

About immunostaining.info

Immunostaining.info is the website that offers you detailed information about IHC protocol. It is a small part of Boster Bio that gives you complete knowledge about IHC principle and IHC protocol. IHC principle is the method of fixation, tissue sectioning, paraffin embedding, inactivation and blocking, antigen retrieval, detection and so on. You will also study complete guide of IHC Sample preparation guide and IHC protocol. For more details visit our website https://immunostaining.info

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