Immunocytochemistry vs. Immunohistochemistry

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're going to be comparing and contrasting two staining techniques, immunocytochemistry and immunohistochemistry. Here, we'll learn about what each of these techniques are used for and how they differ.

What Are Staining Techniques?

Many of us enjoy taking our dog out for a walk after work, but in the fall and winter it can be very dark out. One solution is to tag your pet with a small light on their color or leash. This allows you to see where your pet is as you go out for your evening stroll. Without the light, it would be hard to see what's going on during the walk.

Similarly, the inner workings of cells are essential for understanding disease states, but can be impossible to visualize even with high powered microscopes. Luckily, scientists have a method for tagging proteins of interest, just like you might tag your dog for a walk. These methods are called staining techniques, and there are two main types: immunohistochemistry and immunocytochemistry. Immunohistochemistry stains tissue sections, whereas immunocytochemistry stains single layers of cells grown in culture or from a patient sample. Today we're going to look at the main differences between these two staining types.

Sample Source

One of the most important differences between immunohistochemistry and immunocytochemistry is the source of the sample. Histology is the study of tissue architecture, so we can extrapolate that immunohistochemistry uses tissue sections as the starting sample. And the prefix 'cyto' means cell, so that tells us that immunocytochemistry uses a single layer of cells, or individual cells, as the starting sample.

Although tissues are indeed made of cells, immunohistochemistry preserves the original architecture of the tissue sample. The cells are arranged how they are in the body tissues, including the extracellular components like proteins and carbohydrates.

In immunocytochemistry, cells are grown in a single layer and lack the properties of an entire tissue. This allows researchers to zoom in on the properties of single cells, rather than looking at the entirety of the tissue.


To start either procedure, both samples need to be fixed with a fixative agent, such as formaldehyde. This preserves the structure of the cell or tissue. But tissues need additional steps before they can start immunohistochemistry.

Tissues must be embedded in paraffin wax to preserve their structure prior to being sectioned and mounted. However, the paraffin interferes with the staining procedure and thus must be removed after the samples are mounted, but before the staining starts. This processing can sometimes cause the intended targets for the staining to become obscured, so immunohistochemistry samples must undergo another process called antigen retrieval to re-expose the target proteins.

Cells have different challenges for preparation prior to immunocytochemistry. Individual cells used during immunocytochemistry must be permeabilized to open up the cell membrane and allow antibodies into the cell during staining. Since the tissue sections are already cut, they skip this step. After these preparations, both samples are ready for staining.


Traditionally, immunohistochemistry uses chromogens for their staining method. First, samples are treated with an antibody that is a match for the target protein. Then, a second antibody is used that matches the first antibody. The secondary antibody carries an enzyme that will convert a chemical into an insoluble pigment. The pigment falls near the location of the antibodies, thus identifying the protein.

Chromogen staining of a smooth muscle cell tumor using immunohistochemistry

Immunocytochemistry typically uses fluorophores, fluorescent molecules that are bound to the secondary antibody. First, a primary antibody is incubated with the sample that will bind to the target protein. Then, the fluorescent labeled secondary antibody is added, which binds to the primary antibody. When a special light source is applied, the fluorophore lights up, allowing researchers to locate the protein of interest.

A cancer cell line stained in red and cyan for cytoskeletal proteins and blue for DNA

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