Immunohistochemistry

Also known as:

IHC

Samples needed

Fixed cells or tissue on a slide

Controls

Frequently, in addition to antibodies for the protein of interest, cells are also stained with hematoxylin and eosin (H&E stain). Hematoxylin stains nuclei purple, and eosin stains cytoplasm and extracellular matrix pink. Almost all histology samples, even those not subjected to IHC, are stained with H&E.

As for IF, often times, other controls for IHC are not shown in a published work. However, if a researcher is using new IHC antibodies for the first time, several controls are useful. A positive control that contains the protein of interest tells the researcher if the antibody works well in IHC and/or if the conditions used for staining are optimal. A control without the primary antibody ensures that the secondary antibody is binding specifically to the primary antibody and not other proteins.

Method

Many IHC samples come from a larger piece of tissue. Immediately upon collection, the tissue should be fixed, frequently in formalin. The tissue is embedded in wax, then sectioned, or cut into very, very thin slices, around a few µm thick. The section is mounted on a glass slide. When a researcher is ready to perform IHC, the wax is removed from the sample and antigen retrieval is performed, often by heating. The sample is then blocked, often with milk protein, to discourage any non-specific binding of antibodies, and also any endogenous peroxidase activity is blocked. Next, the sample is incubated in a primary antibody, which specifically binds to the protein of interest. Several different methods can be used at this point, but all result in a colored substance being deposited on the sample only at the locations were the protein of interest is located.  Finally, the sample is counterstained with H&E.