Immunohistochemistry

Immunohistochemistry is a form of immunostaining. It involves the process of selectively identifying antigens in cells and tissue, by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Albert Hewett Coons, Ernest Berliner, Norman Jones and Hugh J Creech was the first to develop immunofluorescence in 1941. This led to the later development of immunohistochemistry.

Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. In some cancer cells certain tumor antigens are expressed which make it possible to detect. Immunohistochemistry is also widely used in basic research, to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.

Immunohistochemistry can be performed on tissue that has been fixed and embedded in paraffin, but also cryopreservated (frozen) tissue. Based on the way the tissue is preserved, there are different steps to prepare the tissue for immunohistochemistry, but the general method includes proper fixation, antigen retrieval, incubation with primary antibody, then incubation with secondary antibody.

Fixation of the tissue is important to preserve the tissue and maintaining cellular morphology. The fixation formula, ratio of fixative to tissue and time in the fixative, will affect the result. The fixation solution (fixative) is often 10% neutral buffer formalin. Normal fixation time is 24 hours in room temperature. The ratio of fixative to tissue ranges from 1:1 to 1:20. After the tissue is fixed it can be embedded in paraffin wax.

For frozen sections, fixation is usually performed after sectioning if not new antibodies are going to be tested. Then acetone or formalin can be used.

Sectioning of the tissue sample is done using a microtome. For paraffin embedded tissue 4 μm is normal thickness, and for frozen sections 4 – 6 μm. The thickness of the sliced sections matters, and is an important factor in immunohistochemistry. If you compare a section of brain tissue measuring 4 μm with a section measuring 7 μm, some of what you see in the 7 μm thick section might be lacking in the 4 μm section. This shows the importance of detailed methods related to this methodology. The paraffin embedded tissues should be deparaffinized to remove all the paraffin on and around the tissue sample in xylene or a good substitute, followed by alcohol.

Antigen retrieval is required to make the epitopes accessible for immunohistochemical staining for most formalin fixed tissue section. The epitopes are the binding sites for antibodies used to visualize the targeted antigen which may be masked due to the fixation. Fixation of the tissue may cause formation of methylene bridges or crosslinking of amino groups, so that the epitopes no longer are available. Antigen retrieval can restore the masked antigenicity, possibly by breaking down the crosslinks caused by fixation. The most common way to perform antigen retrieval is by using high-temperature heating while soaking the slides in a buffer solution. This can be done in different ways, for example by using microwave oven, autoclaves, heating plates or water baths. For frozen sections, antigen retrieval is generally not necessary, but for frozen sections that have been fixed in acetone or formalin, antigen retrieval can improve the immunohistochemical signal.

Non-specific binding of antibodies can cause background staining. Although antibodies bind to specific epitopes, they may also partially or weakly bind to sites on nonspecific proteins that are similar to the binding site on the target protein. By incubating the tissue with normal serum isolated from the species which the secondary antibody was produced, the background staining can be reduced. It is also possible to use commercially available universal blocking buffers. Other common blocking buffers include normal serum, non-fat dry milk, BSA, or gelatin. Endogenous enzyme activity may also cause background staining but can be reduced if the tissue is treated with hydrogen peroxide.