Immuno-Histo-Chemistry (IHC) is an immunostaining technique. These methods include the use of antibodies that attach to their antigens in situ and emit signals to enable to detect them. In Immunohistochemistry, the antigens are studied in cell tissues and precisely localized. This application is continuously developing thanks to imagery technology evolution as well as automation. Moreover, the image analysis tools enable more data collection than ever.
The first step of IHC experiment is the preparation of the sample. It may vary on the tissue and the experiment goal. Studied tissues are typically embedded in paraffin wax prior to sectioning. They are then sliced at typical thickness of 3µm to 5µm. The slices are then fixed on slides and dehydrated. As non-specific binding of the antibody may happen, the cells are incubated with blocking buffers such as milk. This would reduce signal background noise. The labeling step might be optimized for each experiment. A primary antibody is directed to its antigen located in the sample. After a washing step, secondary antibodies are bound to the primary antibodies. They carry a reporter molecule. These molecules may be chromogenic or fluorescent depending on the chosen detection method. They will emit a signal accordingly, enabling to locate the antibodies and thus, the targeted proteins in the fixed cells. Counterstains are also often applied to detect the whole cell or common biomolecules to create a contrast with the reporter molecule staining.
Applications of Immunohistochemistry
Immunohistochemistry may be used in many research areas. Drug and disease regulation may be followed with IHC. In cancer research or diagnostic, a tumor may be identified as malignant or not, and its development stage or origin cell type asserted. This can be done by targeting cancer biomarkers. Other applications include the study of pathologies and developmental biology, cell cycles and wound healing for example.