Immunohistochemistry

Overview



Scientists in various fields of biology need ways to look at tissues in depth. If you look at a piece of blood vessel under a microscope, you probably will not see anything of note. We have a variety of stains that can help visualize different structures, but we can gain the most molecular detail using a technique called immunohistochemistry (IHC).

IHC is based on the principle that antibodies bind selectively to certain proteins. These antibodies are applied to a tissue to detect primary antigens. Then you will detect where the antibody bound.

How Do You Perform IHC?



IHC starts with a tissue taken from an animal or human sample. Typically this sample is embedded in a wax like paraffin before being sliced extremely thin. These thin slices are adhered to a slide, and then the tissue is fixed and permeabilized. Fixation refers to the application of formaldehyde to hold proteins in place within cells. This prevents them from moving around and from diffusing completely out of the cell. Permeabilization is the use of a detergent to dissolve parts of the cell membrane.

After slide preparation, a solution of antibody will be applied. Antibodies are typically grown and purified either from special cell culture or from animals. They detect a specific protein and bind it tightly. After you wash off the excess antibody, you will apply a second antibody solution. This secondary antibody detects the first antibody. The important part of the secondary antibody is the addition of a reporter molecule. In IHC, the typical reporter is alkaline phosphatase or a fluorescent dye. Now you have a protein bound to an antibody bound to another antibody. To facilitate detection, you add a solution that contains the alkaline phosphatase substrate diaminobenzidine. This component reacts with the alkaline phosphatase to form a brown product. If your secondary antibody is attached to a fluorescent dye, detection is as simple as shining ultraviolet light and reading the result using a camera or microscope.

Why Is IHC Used?



IHC is a powerful tool in the arsenal of a cell biologist. It enables you to determine protein amount or protein location within the cell at any given time. This enables you to ask a number of questions that are impossible to answer with many other techniques. Another common application for IHC is the analysis of tumor samples. It can be used in this context to determine whether molecular markers of certain cancers are present. It can also be used to determine prognosis for patients. In these different ways IHC fills a very special and important niche in many different fields of biology.