Immunohistochemistry
- One of the most common applications of immunostaining, used to identify certain tissue
pathologies, such as cancer
- Immunohistochemistry involves the process of selectively identifying antigens in the cells of
a tissue section, by exploiting the principle of antibody-antigen binding in biological tissue
- Immunohistochemistry helps clinicians to identify abnormal cells, such as cancerous cells
Process:
- Tissue fixation – this step is important to maintain tissue structure and retain antigenicity of
the tissue. There are several fixation methods which can be used. For example, if the focus
is on antigen expression but tissue morphology is not too important, snap-frozen or acetone
fixation would be used. If the focus is on tissue morphology, formalin-fixed and paraffin-
embedded (FFPE) would be used
- Antigen retrieval – if using the FFPE method, sample should be pre-treated with antigen
retrieval agents. These improve antigen expression by breaking down formalin-induced
antigen cross links. Proteolytic enzymes can also be used to break down these cross-links.
- Blocking – this is required to stop false-positive staining. In immunohistochemistry, both
endogenous enzymes and antibodies need to be blocked, to prevent them reacting and
affecting immunostaining
- Antibody labelling and visualization – this can be done either directly, attaching a label
directly via covalent bonding to the primary antibody, or indirectly, incubating the antibody
twice before binding
- One of the most common applications of immunostaining, used to identify certain tissue
pathologies, such as cancer
- Immunohistochemistry involves the process of selectively identifying antigens in the cells of
a tissue section, by exploiting the principle of antibody-antigen binding in biological tissue
- Immunohistochemistry helps clinicians to identify abnormal cells, such as cancerous cells
Process:
- Tissue fixation – this step is important to maintain tissue structure and retain antigenicity of
the tissue. There are several fixation methods which can be used. For example, if the focus
is on antigen expression but tissue morphology is not too important, snap-frozen or acetone
fixation would be used. If the focus is on tissue morphology, formalin-fixed and paraffin-
embedded (FFPE) would be used
- Antigen retrieval – if using the FFPE method, sample should be pre-treated with antigen
retrieval agents. These improve antigen expression by breaking down formalin-induced
antigen cross links. Proteolytic enzymes can also be used to break down these cross-links.
- Blocking – this is required to stop false-positive staining. In immunohistochemistry, both
endogenous enzymes and antibodies need to be blocked, to prevent them reacting and
affecting immunostaining
- Antibody labelling and visualization – this can be done either directly, attaching a label
directly via covalent bonding to the primary antibody, or indirectly, incubating the antibody
twice before binding
