Immunohistochemistry (IHC) and Immunofluorescence (IF) Detection Methods
Immunohistochemistry (IHC) is a biochemical method that uses antibodies that bind to specific antigens in a section of tissue. Similarly, immunocytochemistry (ICC) is suitable for identifying antigens in individual cell layers. IHC is commonly used to visualize proteins, carbohydrates, and lipids of interest in both healthy and diseased tissues, such as those found in cancerous tumors. Specific molecular markers are characteristic of cellular events such as proliferation or cell death (apoptosis). IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of biological tissue.
Typically, tissues are fixed, embedded in paraffin, and sectioned for IHC analysis using a microtome. A significant step following the initial tissue preparation is the need for antigen retrieval. It is needed to break the protein cross-links formed during fixation and uncover hidden antigenic sites. Antigen retrieval and pretreatment conditions must be determined empirically, as the accessibility of the antigen epitope may vary significantly depending on numerous biological factors. For example, some antigens may require a more aggressive pretreatment in order to “unmask” their antibody-binding epitopes, while pretreatment may not be required at all for some antigens. Two common antigen retrieval pretreatment methods include Heat-Induced Epitope Retrieval (HIER) where a heat source is used in conjunction with buffers and enzymes (proteinase K). The second method, Proteolytic-Induced Epitope Retrieval (PIER) is another antigen retrieval method where various proteases may be used, such as proteinase k, trypsin, chymotrypsin or pepsin.
Following tissue preparation and antigen retrieval pretreatment, visualizing the antibody-antigen interaction can be accomplished in a number of ways. One common method uses a primary antibody that is conjugated to an enzyme, such as horseradish peroxidase or alkaline phosphatase, that catalyzes a color-producing reaction. Alternatively, the immunofluorescence (IF) method uses an antibody that is tagged to a fluorophore, such as fluorescein, rhodamine, or Alexa Fluor. Another option is to use an unlabeled primary antibody, with indirect antigen detection by a labeled secondary antibody or more complex detection systems. In this case, the optimal titer of both the primary and secondary antibody should be determined for each assay.