Immunohistochemistry (IHC) is a technique for localizing and detection of specific proteins in a tissue section by exploiting the ability of antibodies to bind specifically to antigens. Immunocytochemistry (ICC) is a closely related technique where proteins are localized within cells (usually from cell culture) rather than tissues.
Tissue preparation is critical to ensure preservation of cellular morphology and epitope availability. IHC samples are commonly fixed in paraformaldehyde then embedded in paraffin and sectioned. In some cases, epitope retrieval techniques are used to improve staining for antigens that are affected by para-formaldehyde. Detection of sensitive antigens may require preservation of tissues and cells by flash freezing. Freezing maintains epitope activity, but may compromise sample morphology.
Incubation of the sample with a primary antibody is used to localize the protein of interest. The primary antibody may be monoclonal or polyclonal. Monoclonal antibodies interact with a single epitope on the target, and are generally considered to provide higher specificity of detection. Polyclonal antibodies recognize multiple epitopes, and in many cases provide better sensitivity. Usually, primary antibodies are unlabeled.
The secondary antibody is directed against the species of the primary antibody and typically carries a label for detection. The label may be a fluorophore, enzyme or hapten:
- Fluorophore labeled secondary antibody may be detected directly if the protein of interest is relatively abundant. This technique is known as immunofluorescence (IF).
- Enzyme labels used for detection include horseradish peroxidase (HRP) and alkaline phosphatase (AP). The enzymes react with a substrate in order to produce a signal. DAB and BCIP/NBT are commonly used substrates for chromogenic detection in bright field microscopy.
Note: It is critical to quench any endogenous peroxidases or phosphatases that are present in the sample if these enzymes are used for detection.
- Hapten labels allow introduction of an additional round of detection reagents (as streptavidin or antibody conjugates), often for higher sensitivity. Common haptens include biotin, fluorescein and DNP.
Note: It is critical to quench any endogenous biotin activity if present in the sample in case biotin is used for detection.
TSA for IHC, ICC and IF
Researchers have used Tyramide Signal Amplification (TSA™) and TSA Plus for signal amplification in a wide variety of sample preparations, including cultured cells, formalin fixed paraffin and methacrylate embedded sections, frozen tissue sections and free floating sections. TSA has been used for signal amplification with a broad range of instruments including brightfield microscopes, standard and confocal fluorescence microscopes, electron microscopes and High Content Screening systems like Operetta®.
TSA amplification reagent is a substrate for HRP that is easily introduced into standard protocols with a short incubation step of less than 10 minutes. HRP converts the TSA reagent to a highly reactive free radical that binds covalently to electron-rich amino acids like tyrosine and tryptophan immediately proximal to the target. The TSA reagent is labelled with a fluorophore for immediate detection or with a hapten for chromogenic, fluorescent or EM detection with additional reagents.
The result is vastly improved sensitivity and outstanding resolution for subcellular localization of low abundance proteins. TSA Plus provides 10-20 times higher sensitivity than standard TSA, and typically >1000 fold higher sensitivity than other methods, making it ideal for detection of weakly expressed targets.
Visit the TSA references page to find out how scientists across the globe are using Tyramide Signal Amplification (TSA) for immunohistochemistry techniques.
IHC detection of CD3 antigen in serial sections of paraffin-embedded human tonsil
Rabbit anti-CD3 1:400, biotinylated anti-rabbit, ABC,
with DAB chromogenic detection.
TSA biotin: rabbit anti-CD3 1:400, biotinylated anti-rabbit,
SA-HRP, biotinyl tyramide, ABC, with DAB chromogenic detection.