What is IHC?

Immunohistochemistry (IHC) =Immunology +histology + chemistry

• IHC is an application of antibodies to tissue preparation
– for the localization of target antigens
– visualized using a microscope

Definition:
Immunohistochemistry (IHC) is a staining technique, utilizing the
specificity of antibodies to their antigen in cells of a tissue section,
targets fluorescent dyes to specific antigen in cells.

Production of primary reagents

Pivotal reagent in immunohistochemistry and they impact specificity
Two types of abs are used
1. monoclonal antibody
2. polyclonal antibody
 Main Step in Immunohistochemistry (IHC)
1. Tissue fixation
This step is pretty important as it maintains tissue structure and
retains antigenicity (the availability of the antigens/proteins to
be detected by antibodies). The fixation method you use
depends on the tissue type you are using and your
individual experimental requirements.
 Snap frozen and acetone-fixed tissue ---------- good
antigenic expression
 formalin-fixed and paraffin-embedded (FFPE) ------- clear
morphology
 2. Antigen Retrieval

Antigen retrieval is the most important step in immunohistochemistry.

Actually most formalin fixed tissues required an antigen retrieval step
before immunohistochemical staining.
In formalin fixed tissue, Methylene Bridge formed during fixation which covers
the antibody binding sites on tissue. Antigen retrieval reagents break the
protein crosslinks formed by formalin fixation and there by uncover hidden
antigenic sites.

 different methods
The two methods for antigen retrieval are heat induced
1. epitope retrieval (HIER)
2. Enzymatic retrieval.
3. Blocking
Blocking is essential for preventing
Non-specific binding of antibodies
Or other reagents to the tissue.
4. Antibody labeling and visualization
IHC staining can be set up in one
of two ways – as an indirect assay,
or using direct
Labeling.