Last revised by Frank Gaillard on 7 Aug 2023

Immunohistochemistry is a method of assessing histology with the use of antibodies to specific antigens. It is complementary to the older technique of chemical staining of tissues (such as the very commonly used hematoxylin and eosin stain).

There are many variations on exactly how immunohistochemistry is performed and it is outside the scope of this article to cover any in great detail. Still, broadly speaking they can be divided into:

  • immunofluorescence, where the antibody is tagged with a compound that fluoresces; and

  • chromogenic immunohistochemistry, where an enzyme is linked to the antibody that catalyzes a chemical reaction resulting in the deposition of visible pigment (typically brown)

Both processes employ antibodies to specific antigens (e.g. cellular proteins like GFAP or EMA). There are now a significant number of antibodies, and typically multiple antibodies (a panel) will be used based on the differential diagnosis suggested by the patient's history and standard H&E examination of the sample. 

Direct vs indirect

Immunohistochemistry can also be divided into direct and indirect methods.

Direct immunohistochemistry is when the antibody that links to the antigen is also the one with the ability to be visualized (whether it be by fluorescence or pigment).

Indirect immunohistochemistry, on the other hand, has two antibodies. The first (primary antibody) binds to the desired antigen and the second one (secondary antibody) has the fluorescent or enzyme attached and it binds to the primary antibody. The benefit of this approach is that it can amplify the signal (multiple secondary antibodies can bind to the primary antibody) and it can standardize the subsequent steps.

Indirect methods are most commonly used.

Slide preparation

Immunohistochemistry is a complicated process that require a number of sequential steps in order to achieve quality results. Chromogenic immunohistochemistry typically includes the following steps 2:

  1. antigen retrieval

  2. primary antibody that binds to the antigen

  3. secondary antibody that binds the primary antibody

  4. application of a chromogen localize the primary antibody

  5. counterstain to help give context to the location of the primary antibodies

Antigen retrieval

Due to tissue properties as well as due to slide preparation and fixation techniques most antibodies are not readily available for binding to the primary antibody. Therefore, a variety of processes, collectively known as antigen retrieval, are employed to prepare the sample. This includes physical measures such as heat or exposure to ultrasound, or chemical agents such as denaturants or enzymes. These are often performed concurrently (e.g. an denaturant to break down proteins crosslinks will be applied at the same time as heat) 2.

Primary antibody

The primary antibody is the one that is specific to the antigen that is being investigated.

Secondary antibody

The secondary antibody is used to bind to the primary antibody and has an attached label that may be fluorescent or an enzyme that catylises a visible color change in subsequent steps. The application of secondary antibodies also allows the amplification of signal by multiple secondary antibodies binding to each primary antibody.

Chromogen application

In the setting of chromogenic immunohistochemistry a chemical (chromogenic substrate, e.g. diaminobenzidine) that the label on the secondary antibody (e.g. horseradish peroxidase) converts to a visible stain is used.


Generally, chromogenic immunohistochemistry is combined with a counter-stain for context (e.g. hematoxylin to stain cell nuclei blue). This allows the location of the antibodies to be identified relative to tissue structures.

Internal control

Often slides will have a sample of some other tissue known to react with the antibody to act as an internal control.

ADVERTISEMENT: Supporters see fewer/no ads

Cases and figures

  • Figure 1
    Drag here to reorder.
  • Figure 2
    Drag here to reorder.