Introduction
Immunohistochemistry
Immunohistochemistry (IHC) is a powerful technique that allows for the visualisation of specific proteins (or other molecules) within cells and tissues. It relies on the highly specific interaction between an antigen and its antibody. This technique is widely used in research, pathology, and clinical diagnostics to identify specific proteins, understand their roles, diagnose diseases, and predict treatment responses.
Antibodies: The Key to Specificity
IHC requires antibodies against the protein of interest. These antibodies can be either polyclonal, meaning they are a mixture of antibodies that bind to different regions of the protein, or monoclonal, meaning they are a single type of antibody that binds to a specific region of the protein. Monoclonal antibodies are often preferred as they are highly specific and bind strongly to the target protein, reducing non-specific binding to other similar proteins.
Production of Antibodies for IHC
The production of antibodies for IHC involves injecting the purified protein of interest into an animal of a different species. For example, to produce antibodies against a human protein, the protein would be injected into a rabbit or a goat. This injection triggers the animal's immune system to produce antibodies against the foreign protein.
Direct and Indirect Methods of IHC
There are two main methods of IHC:
Direct IHC: This method uses an antibody that is directly tagged with a label, such as a fluorescent compound or an enzyme like peroxidase. This labelled antibody is incubated with the tissue section, allowing it to bind to the target protein. The section is then washed, processed according to the label, and examined under a microscope to visualise the location of the target protein.
Indirect IHC: This method uses two antibodies and involves more washing steps. A primary antibody specific to the target protein, but unlabelled, is applied first. A secondary antibody, labelled with a detectable tag, is then applied. This secondary antibody is designed to bind to the primary antibody, amplifying the signal because multiple secondary antibodies can bind to a single primary antibody
. For example, if the primary antibody was produced in a mouse, the secondary antibody would be produced in a rabbit or goat and would be specific to mouse antibodies.
Detection Methods in IHC