Method of the Month: Immunohistochemistry
March 2023
Background
During research, scientists will often take tissue samples to analyze under a microscope. They look for distinct differences between the cancerous or diseased cells and the healthy cells. However, there are cases where diseased and healthy tissue looks similar under a microscope. It is even possible to have two diseases or sub-diseases that will appear the same under a microscope. This makes it incredibly difficult for doctors to diagnose and treat patients. Fortunately, there are methods of analyzing tissue that resolve this dilemma.
Back in the early 1940s, scientists, Coons et al., released findings from their study using a method called Immunohistochemistry. Immunohistochemistry (IHC) is the process of using specific binding antibodies (large counteracting proteins) to detect antigens (proteins that induce an immune response) on tissue that scientists are trying to localize. Antibody proteins, built in a ‘Y’ shaped manner, have the ability to target specific antigens, which lie on the surface of every cell, because of their antigen binding site. The chemical interactions between antibodies and antigens at the binding sites are the driving force of this method.
What makes Immunohistochemistry unique is that it incorporates the preservation of anatomical and structural features of a tissue sample. Because of its detail, this method can be used to identify cancers like mesothelioma and lymphoma.
Method
Immunohistochemistry involves the process of selectively identifying antigens in cells of a tissue sample by taking advantage of the idea that antibodies will bind specifically to antigens in tissues. Antibody performance by IHC is highly dependent on the protocols and reagents used during the antigen retrieval, blocking, and detection process. The goal is to use antibodies to stain and analyze protein expression while maintaining cell structure and cell characteristics.
The steps of immunohistochemistry are as follows: deparaffinization, rehydration, antigen retrieval, blocking, primary antibody, secondary antibody, detection, and counterstain.
To begin, scientists must first prepare a tissue sample. They will slice a tissue sample and place it on a microscope slide for deparaffinization. Deparaffinization is the process of removing paraffin, a waxy substance, from the sample (only necessary if the sample was derived from a paraffinized tissue used to preserve it). This is done by repeatedly immersing the slide in xylene, which also dehydrates the sample. To rehydrate, the next step of the process, the sample is immersed repeatedly in deionized water.
Antigen retrieval, meant to remove crosslinks between proteins and unwind them, can be done using several different methods, the most common being HIAR, heat-mediated antigen retrieval. This is done by immersing the slides in a retrieval buffer and heating them using a microwave. The retrieval buffer is meant to remove chemical modifications, keep the proteins unwound, and prepare the slide for blocking.
Blocking, meant to ensure specific binding, is done by adding drops of a universal blocking buffer to the slide for a period of time and then rinsing it with deionized water. The universal blocker is a mix of diluted solutions of various proteins which coat the sample with excess protein that can attract any unbound primary or secondary antibody in the solution and prevent non-specific binding of the antibodies to the tissue section. Specific binding is critically important for the success of the IHC method.
Next, the slides will be exposed to concentrated solutions of primary antibodies which bind to the antigens on the cells doctors are trying to identify. The slide is then incubated and washed for a period of time. This process will be repeated using a solution of secondary antibodies, which will bind to the primary antibodies from the previous step. Secondary antibodies will have a tag or marker, attached in the next step, which is what gives the cells we want to identify color under the microscope.
Detection, also called indirect detection and amplification, is the step of attaching tags or markers to the secondary antibodies from the previous step. This can be done in two ways: chromogenic amplification (the addition of enzymatic substrates to the slide) or fluorescent amplification (the addition of fluorescent dyes to the slide).
This image displays what should be the result of the successful completion of the primary and secondary antibody steps as well as the detection step.
Counterstaining is the final step of the process, which is meant to simply dye the surrounding cells and their nuclei for a contrast in the image when looking under the microscope. After this, the slides can be mounted and examined.
The end result of this process should come out as a beautiful, bright image of a tissue sample with a highlight on the diseased cells scientists were trying to identify. As previously mentioned, specific binding is the key to success in immunohistochemistry. If the primary antibodies bind to the wrong antigen or protein on the cell membrane, then the image produced will not be accurate to the reality of the issue. This is why the blocking step is so important. Doctors need to be able to accurately visualize what is going on inside a patient, and Immunohistochemistry is one way this is possible.
For more information on Immunohistochemistry, check out the following links!
