Imunohistochemistry , also known as IHC is a combination of histology and immunology . it consists of three important component : antibodies , signal, generation systems for tracking and the solid phase in which the reactions take place.

It is used in histology to detect the presence of specific protein marker that can assist with accurate tumor classification and diagnosis. This guide illustrates the basic steps used to create an IHC stain.

The word is made up of two parts , histo meaning tissue and immuno which is related to immune system treatment.

In this method the presence of absence of a specific antigen in the tissue can be investigated using an antibody that specifically binds to the antigen.. this connection can be direct (single phase) or indirect (2 phase).

Antibodies & antigens work like a lock and key, in this mechanism there is a specific antigen attacks your body, the antibody attaches to it with destroy it , thus prevent it from reproducing , spreading and further damaging by doing this.

Any substance that induces the immune system to produce antibodies against it is called an antigen. Any foreign invaders, such as pathogens (bacteria and viruses), chemicals, toxins, and pollens, can be antigens. Under pathological conditions, normal cellular proteins can become self-antigens.


IHC of human kidney tissue labeled with rabbit monoclonal anti-ZO1 (ab221547). Antigen recovery by heat was performed using Tris / EDTA buffer, pH 9.


n general, antigens are composed of proteins, peptides, and polysaccharides. Any portion of bacteria or viruses, such as surface protein, coat, capsule, toxins, and cell wall, can serve as antigens.



IHC image

Primary Antibody

The first stage of IHC is the application of a primary antibody that binds specifically to the target antigen.

There are two main types of antibody, polyclonal and monoclonal. Polyclonal antibodies have an affinity with, and bind to, multiple epitopes (or parts) or the target antigen, and as such are more prone to cross-react to non-target antigens.

Monoclonal antibodies have an affinity to only one epitope and tend to produce, cleaner, more specific staining but are less sensitive or intense.

Secondary Antibody

Next, secondary antibodies bind to the primary antibody. This is known as indirect IHC. It is now commonly used as multiple secondaries can bind to a single primary to amplify the staining intensity.


ایمونو هیستوشیمی
IHC image of neun fluorescent in sections of mouse cerebellum tissue





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