Immunohistochemistry (IHC) uses antibodies binding specifically to antigens in biological tissues to visualize proteins, carbohydrates, and lipids of interest. IHC is an excellent detection technique and has the tremendous advantage of being able to show exactly where a given protein is located within the tissue examined. Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristic of particular 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 a biological tissue. It is also an effective way to examine the tissues .This has made it a widely-used technique in the neurosciences, enabling researchers to examine protein expression within specific brain structures.
Rabbit anti-α-internexin (Catalogue Number AB5354) and Mouse anti-NF-L (Catalogue Number MAB1615) staining of cultured rat neurona. Mature neurona are green. (NF-L positive) and neuronal progenitor cells are red (α-internexin positive).
Tissues are fixed, embedded, sectioned in a similar was as for traditional histology staining. One main difference is the need for antigen retrieval. It is needed to break the protein cross-links formed during the fixation and uncover hidden antigenic sites.
Heat Induced Epitope Retrieval (HIER) is a commonly used antigen retrieval method. A microwave oven, a pressure cooker or an autoclave are commonly used sources of heat, in conjunction with buffers and enzymes (proteinase K).
Proteolytic Induced Epitope Retrieval (PIER) is another antigen retrieval method. Various proteases may be used, such as proteinase k, trypsin, chymotrypsin or pepsin.
Triton X-100 is used to improve antibody penetration. Visualizing the antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, the primary antibody is conjugated to an enzyme, such as horseradish peroxidase or alkaline phosphatase, that can catalyzes a color-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein, rhodamine or Alexa Fluor. Colloidal gold may also be used. Another option is to use an unlabeled primary antibody, with detection by a labeled secondary antibody or more complex detection system. In this case, the optimal titer of both the primary and secondary antibody should be determined for each batch.