Immunohistochemistry (IHC)


What is immunohistochemistry?

Immunohistochemistry (often abbreviated as IHC) is a laboratory test that pathologists use to detect specific proteins, also known as antigens, within cells in a tissue sample. By highlighting these proteins, pathologists can gain a better understanding of the types of cells present, their origins, and their behavior. This information enables doctors to make accurate diagnoses, predict the progression of a disease, and select the most effective treatments.

How does immunohistochemistry work?

Immunohistochemistry utilizes specialized chemicals known as antibodies. These antibodies can recognize and attach themselves to specific proteins inside the tissue sample. Once an antibody binds to its targeted protein, pathologists can make it visible under the microscope by adding a dye or fluorescent marker. This makes it easy to visualize the location of the protein within the cells.

What are the steps involved in immunohistochemistry?

  1. Sample preparation: First, tissue samples are obtained through a biopsy or surgery. To preserve them, specimens are treated with a special solution, usually formalin, and then embedded in a wax-like substance called paraffin.

  2. Sectioning: The preserved tissue is cut into very thin slices (approximately 4-5 micrometers thick) and placed on glass microscope slides, where it is prepared for staining.

  3. Deparaffinization and rehydration: The wax (paraffin) is removed using special chemicals, allowing the tissue to be stained effectively.

  4. Antigen retrieval: Sometimes, proteins (antigens) become hidden during tissue preparation. Pathologists treat the tissue with heat or special enzymes to re-expose these proteins, making them easier for antibodies to find.

  5. Blocking: To ensure the antibodies only attach to their intended proteins, pathologists use special protein solutions to block other unwanted binding sites.

  6. Primary antibody incubation: The slides are covered with a primary antibody that specifically attaches to the protein of interest.

  7. Detection: After washing away any excess primary antibody, a secondary antibody is applied. This second antibody sticks to the primary antibody and carries either an enzyme or a fluorescent marker. When activated, it produces color or fluorescent light, revealing the exact location of the target protein.

  8. Counterstaining: To help pathologists see the tissue structure clearly, a mild background stain (often hematoxylin, which colors cell nuclei blue) is added.

  9. Mounting and visualization: Finally, the slides are covered with a thin coverslip and examined under a microscope. Pathologists note the patterns, intensity, and location of staining to interpret the results.

Why is immunohistochemistry important?

Immunohistochemistry is extremely valuable because it helps pathologists diagnose diseases accurately. It can distinguish between different types of cancer cells, identify infections, and differentiate diseases that look similar under the microscope. Additionally, by identifying specific proteins, doctors can determine the most effective treatment options, including targeted therapies.

What do different staining patterns mean?

When pathologists examine immunohistochemistry slides, they closely examine the distribution of proteins within cells. There are three main patterns: nuclear expression, cytoplasmic expression, and membranous expression. Each is explained in more detail below.

Immunohistochemistry

Nuclear expression

Nuclear expression (also known as nuclear reactivity) refers to the staining that is located in the nucleus of the cell, where genetic material, such as DNA, is stored. Proteins found in the nucleus are often involved in regulating how cells grow and behave. For example, the estrogen receptor (ER), a protein important in breast cancer treatment decisions, appears as nuclear staining. Identifying nuclear staining helps doctors diagnose certain cancers and determine appropriate treatments, such as hormone therapy.

Cytoplasmic expression

Cytoplasmic expression (also known as cytoplasmic reactivity) occurs in the cytoplasm, the area around the cell’s nucleus that contains many important structures and enzymes. Proteins found here typically play a role in metabolism, provide structural support, or facilitate internal signaling. An example of cytoplasmic staining is the presence of cytokeratins—proteins found in cells lining many parts of the body. This type of staining helps identify the origin of cells, diagnose specific types of cancer, or detect infections.

Membranous expression

Membranous expression (also known as membrane reactivity) highlights proteins located on the cell’s surface or membrane, which helps cells communicate with each other and their surroundings. A well-known example is the HER2 protein in certain breast cancers, which is characterized by a distinct membranous staining pattern. Identifying membranous proteins is crucial because it can guide targeted therapies, helping doctors select treatments tailored to the patient’s specific type of cancer.

Understanding these patterns of expression helps pathologists provide accurate diagnoses, better predict how diseases may progress, and recommend effective treatments.

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