Immunohistochemistry

Sometimes it is difficult to classify a tumour type based on its morphology or on the results of various tinctorial staining methods. In recent years, researchers have shown that many types of tumour display a unique combination of cell surface markers which are the result of faulty protein expression or the expression of proteins whose only known function is in the control or regulation of tumours. These marker proteins are, of course, macro-molecules and are invisible to the naked eye and even to light microscopes. It is with a great deal of difficulty and a very high magnification that shadows of these molecules may be observed using an electron microscope. Preparation of electron microscopy sample is both hazardous and expensive however. In many laboratories, the solution has been immunohistochemistry. This technique emerged in the 1980's and has gone from strength to strength with more new antibodies being made available every year.

As I have just hinted, the method is not based on the same type of chemical reactions as the standard tinctorial methods. This method uses immunological reactions. A primary antibody is raised against the protein to be marked and a diluted solution of this antibody is used to treat the tissue section. Later, a secondary antibody, directed against the primary antibody is used. The secondary antibody is usually bound to a protein called biotin. Biotin can bind up to four avidin molecules and the addition of avidin is the next step. As you can imagine, on a molecular level, the complex we are building is pretty large.

The avidin molecules are usually linked to an enzyme called HRP (Horseradish Peroxidase). Finally, the enzyme is given its substrate, peroxide, which is used to activate a very nasty (carcinogenic) compound called DAB (diaminobenzidine). The end reaction is brown. Counterstained cells are blue - see the picture to the right which shows bladder tissue "stained" with an antibody to TGF-ß (Tumour Growth Factor Beta).

Other methods for immunohistochemistry also exist but as this site is not intended to be a technical manual they will not be discussed here.