Immunocytochemistry: Definition, Uses & Techniques

Instructor: Alexandra Unfried

Alexandra earned her master's degree in nursing education and is currently a hospital supervisor/administrator.

Immunocytochemistry is used as a laboratory technique to identify proteins and peptides. This lesson will discuss the definition, uses, and techniques of immunocytochemistry.

Laboratory Testing

Phil is working in the laboratory and assigned to complete immunocytochemistry testing. Phil makes sure that he has the appropriate equipment at his workspace including several microscopes and his reporters that he will use to stain the samples. He has not worked with immunocytochemistry for several months so he reviews the laboratory manual to make sure he is familiar with the process.

What Is Immunocytochemistry

Immunocytochemistry is a procedure that is done in a laboratory that visualizes proteins and peptides. Samples are obtained from sections of tissues, mouth swabs, blood, or any other type of sample from humans, plants, and animals. Biomolecules or antibodies are applied to samples which react with the protein (antigen) that is being tested, causing a change in color to identify the protein. The antibody is associated with a reporter such as an enzyme, fluorescent dye, or fluorophore. The reporter changes the color of the protein which is seen by using a microscope.

Phil has his equipment and is now taking time to familiarize himself with the samples he is testing. The type of sample being tested determines if there needs to be an incubation period before the staining can begin, which type of reporter will be used, and what microscope is needed for visualization.

Microscope slide sample for viewing
Microcrope slide sample for viewing

Immunocytochemistry Uses

The body is made up of many different types of proteins, peptides, and antigens that help carry out functions of the immune system. Antigens are single molecules that combine to form peptides. Peptides are called proteins when they contain more than fifty amino acids. The identification of proteins in cells is important in recognizing the body's immune response to various antibodies. Antibodies are used to fight infections in the body. Immunocytochemistry helps researchers determine which antibodies will bind to specific antigens and build immunity.

Phil is now ready to start applying immunocytochemistry to his samples. Some samples require direct detection while others require indirect detection. He gathers the samples that are ready after necessary incubation time.

Technique for Immunocytochemistry

There are four basic steps to complete immunocytochemistry testing. The first step is cell seeding where the samples are placed on glass slides or transparent plates. Samples require different incubation times before they can be used. Some samples are used right away while others must set for several hours or even twenty-four hours.

The next step consists of fixation of the cells and then immunostaining the cells. A fixative solution is applied to the cells so that they will not move on the slide. Some slides also need to be permeabilized with another solution in order for the stain to react depending on the type of fixative used. The antibody or immunostain (reporter) is then applied to the slide and later rinsed off to ensure excess antibodies are removed.

The third step uses the microscope to visualize the sample and images are developed. The fourth step involves analyzing the images that are written down as an explanation.

Microscope used in immunocytochemistry
Microscope used in immunocytochemistry

There are different reporters used to identify proteins in the cells. It is important to properly label the slides so information is accurate. The different types of reporters that are used to stain the cells are:

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