Immunohistochemistry: Definition, Uses, Stains & Protocols

Instructor: Amanda Robb

Amanda holds a Masters in Science from Tufts Medical School in Cellular and Molecular Physiology. She has taught high school Biology and Physics for 8 years.

In this lesson, we'll be looking at the technique of immunohistochemistry. Here, we'll explore the applications of this technique in medicine and scientific research, as well as the stains used and protocol for carrying out the procedure.

What Is Immunohistochemistry

Have you ever gotten a cut and decided to clean it with peroxide? What happened? If you could see past the painful burning sensation, you probably also noticed that peroxide bubbles when you place it on your open cut. Why is that? Small proteins called enzymes inside your skin change the peroxide into water and oxygen gas, which creates the bubbles. Some bacteria lack these enzymes and so the toxic peroxide kills them while leaving your cells intact, sterilizing your cut.

There are thousands of enzymes inside our cells, but humans have found a use for enzymes in molecular biology as well. During a process called immunohistochemistry, cells or tissue samples are stained with antibodies attached to enzymes. When a substrate for the enzyme is added, the enzymes catalyze a chemical reaction causing a color change that can be viewed under the microscope. This allows scientists to detect the presence of certain proteins in a sample.


So, what good is this technology? Why would we want to look at something so small as protein? Proteins are the building blocks of the cell. They hold the cell together, create internal structures, allowing for movement, cell division, and carry out all other functions of the cell. Changes to protein structure or function are one of the hallmarks of many diseases.

For example, during Alzheimer's disease, a protein called amyloid beta builds up in brain tissue, which can be seen using immunohistochemistry. Changes in protein expression can help scientists understand what is happening during disease progression, giving them avenues to pursue in finding treatments.

Immunohistochemistry is also useful for diagnosing the progression of cancerous tumors. Tumors tend to develop certain protein markers as they become more malignant. Immunohistochemistry can detect these protein changes and also indicate what tissue type the tumor originally came from and what treatments they may be responsive to.

Immunohistochemistry also has other applications, such as identifying a pathogen during an infection by looking for proteins associated with that particular pathogen, detecting brain trauma, and identifying patients with inherited muscle disorders by detecting the presence of a particular protein variant.


There are two main stains that are used in enzyme-based immunohistochemistry, horseradish peroxidase, and alkaline phosphatase. Fluorescent molecules can also be used during a specialized immunohistochemistry procedure called immunofluorescence.

  1. Horseradish peroxidase is an enzyme that converts a colorless substrate into a colored product in the presence of hydrogen peroxide. The colored product precipitates near the antibody, which can be seen under the microscope indicating the presence of the protein. Different substrates produce different colored products. 3,3'-Diaminobenzidine (DAB) is commonly used and produces a red product.
    A breast cancer tumor stained with horseradish peroxidase in brown and counterstained with blue dye to visualize all cells
    breast cancer IHC

  2. Alkaline phosphatase similarly is an enzyme that converts a colorless substrate into a colored product that precipitates and binds to the tissue where the antibody is located. Alkaline phosphatase typically is used with nitroblue tetrazolium (NBT), which produces a blue precipitate.


1. Fix

The first step is to use a fixative such as formaldehyde to preserve the tissue. This prevents the tissue from decomposing and maintains cell structure.

2. Section and Mount

After the tissue is fixed, most tissue samples are embedded in paraffin wax to preserve the tissue architecture. After samples are paraffin embedded, they can be sectioned for mounting on glass slides that can be viewed under a microscope.

3. Deparaffinization and Antigen Retrieval

Although the paraffin is useful for maintaining structural integrity during the sectioning, it interferes with antibody attachment during immunohistochemistry. Thus, the next step is deparaffinization where paraffin is removed from the samples using a chemical treatment, usually xylene. The fixative can also block antibody binding sites and must be removed using additional chemicals in a process called antigen retrieval.

4. Block

The enzymes horseradish and alkaline phosphatase are both naturally found in tissues, and thus their activity must be quenched before starting the staining procedure by incubating the sample with either hydrogen peroxide for horseradish peroxidase samples or levamisole for alkaline phosphatase samples.

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