IHC Antibodies

Immunohistochemistry (IHC) is a technique that uses antibodies for visualizing targets of interest in tissue samples. The samples may be preserved by formalin-fixation and paraffin-embedding (FFPE), which maintains tissue morphology and allows for long-term storage at ambient temperature. Alternatively, they may be snap-frozen in liquid nitrogen. Freezing is generally preferred when there is a need to conserve enzyme function or protect the antigenicity of sensitive epitopes such as post-translational modifications (PTMs). When FFPE samples are used, the IHC workflow begins with slicing the tissue into thin sections with a microtome and applying these to slides. The sections must then be deparaffinized by immersion in xylene, before being rehydrated with decreasing concentrations of alcohol. Once the sections have been washed with deionized water to remove any last traces of alcohol, epitope retrieval may be necessary. This serves to expose any epitopes that have become masked by formalin fixation and may either be heat-induced (HIER) or based on the use of proteolytic enzymes such as proteinase K, trypsin, or pepsin (PIER). When working with frozen samples, deparaffinization and rehydration are unnecessary, and fixation is only performed once the sections have been applied to the slides. Another key protocol difference between the two preservation methods is that epitope retrieval is less often required for frozen tissue. Notably, when using samples that have been fixed with an alcohol (e.g., methanol) rather than an aldehyde-based fixative (e.g., formalin, formaldehyde, or glutaraldehyde), the epitope retrieval step can be avoided. From this point onwards, the workflows for both FFPE and frozen tissue converge. In both cases, permeabilization is required when detecting intracellular targets, and is followed by blocking to prevent unwanted background signal. Blocking of non-specific antibody binding typically involves incubating the tissue sections in normal serum. Additional blocking steps to consider include mouse-on-mouse blocking when using mouse primary antibodies to probe mouse tissue, and blocking of endogenous peroxidase, phosphatase, or biotin depending on the chosen detection method. When selecting IHC antibodies, researchers must decide whether to perform direct detection with labeled primary antibodies or indirect detection with secondary antibodies. Advantages of direct detection are that it shortens the experimental workflow and allows for multiplexing with antibodies sharing the same host species. It is worth noting that while direct detection was once limited by the availability of labeled primary antibodies, researchers now have the option to buy a carrier-free antibody and label it in-house. A main benefit of indirect detection is that it provides signal amplification, which can increase the likelihood of detecting scarce targets. Researchers must also select which type of readout they wish to use. Chromogenic detection combines enzyme-labeled antibodies with substrates such as 3,3′-diaminobenzidine (DAB) to generate a colored precipitate that can be viewed via standard microscopy. Fluorescent detection instead uses fluorophore-labeled antibodies and requires access to a fluorescence microscope. When choosing between the different approaches, factors to consider include which reagents are available and whether multiplexing is necessary. The final stages of the IHC workflow involve counterstaining (i.e. for nuclei) and mounting prior to visualization and analysis. For accurate results, it is important to include relevant controls, such as positive and negative sample types, unstained sections, and isotype controls.