There are a variety of experimental methods to choose from when using antibodies as a probe to highlight a target of interest. Western blot will reveal protein abundance and behavior, immunocytochemistry allows a look at protein behavior on the cellular level, and flow cytometry has the ability to label and sort hundreds of thousands of cells in no time. However, immunohistochemistry (IHC) is the only method where researchers have the ability to view the spatial localization of a target within a specific tissue. A lot has changed with IHC since its introduction in the mid 1900s, including automated processing machines, multiplexing techniques, advanced image analysis, robustly validated IHC antibodies and more. These tools have increased clinical diagnostic efficacy for cancer (among other disease) and continue to strengthen the role of IHC in the laboratory.
ErbB2/Her2 was detected in immersion fixed paraffin-embedded sections of human breast cancer tissue using Goat Anti-Human ErbB2/Her2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1129) at 1.7 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to epithelial cells. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.
Automated instruments for IHC analysis have become increasingly popular. Such machines are produced by Leica and others, and include automated slide staining, fixation and imaging. Typically these systems are described as either “open” or "closed". Open simply means that you can use antibodies and reagents from any desired vendor, whereas a closed system limits products to one supplier. These IHC stainers save researchers time and increase the reproducibility of their results. Heat fixation is one example of reproducibility that automated slide strainers introduce, as consistent slide heating yields consistent heat induced epitope retrieval. In addition to the above applications, automated tissue segmentation has also emerged, where the tissue section is stained with various antibody markers to perform trained image analysis for segmentation. In the end, there are various models of automated IHC instruments on the market, and it is important to have a thorough understanding of IHC protocols before choosing one that fits your experimental needs.
Multiplexing immunohistochemistry techniques (mIHC) are also becoming more common. One such method is known as serial section, or feature analysis on consecutive tissue sections (FACTS). In this technique, brightfield or fluorescent multiplex staining using primary antibodies is performed on individual tissue sections, the images are analyzed, and a feature extraction aligns the sections for further analysis. When it comes to imaging, confocal laser scanning microscopy is unique in that multiple lasers allow for multispectral detection. Furthermore, a type of next generation immunohistochemistry has also been introduced, where lanthanide metal conjugated primary antibodies are used and tissue sections undergo mass spectrometry.
Learn more about IHC with our handbook.
