Immunohistochemistry (IHC) is a technique that uses antibodies to visualize specific antigens within preserved tissue sections. In contrast to practices like Western blotting, which involve analysis of bulk samples such as tissue lysates, IHC provides valuable information regarding antigen localization and relative expression levels in the context of tissue anatomy.
Sensible interpretation of IHC staining data requires that a multitude of variables are carefully optimized. In this article, MilliporeSigma shares with us their top tips for IHC success and explains how the Human Protein Atlas is a powerful tool being used to validate antibody specificity.
Tissue samples for IHC staining can be either frozen or formalin-fixed paraffin-embedded (FFPE). Advantages of freezing include retention of antigen structure and faster processing times, while disadvantages include a loss of tissue morphology as an artefact of freezing, and the requirement for cold storage. FFPE tissue affords easier handling, viable long-term storage at ambient temperature, and can often be more suitable for IHC, yet a drawback is that cross-linkage of proteins can often necessitate antigen retrieval. The method chosen will be influenced by factors including sample-handling capacity, available cold storage facilities, the need for sample archiving, and antibody compatibility with antigen retrieval.
FFPE samples typically require that an antigen retrieval step is performed prior to IHC staining because the fixation process can often result in antigen masking. Protease-induced epitope retrieval (PIER) involves treating samples with enzymes such as proteinase K, trypsin, or pepsin, but has a relatively low success rate and can damage tissue morphology. Heat-induced epitope retrieval (HIER), often performed with microwaves or water baths, is more popular, but is highly sensitive to timing, temperature, buffer, and pH. Antigen retrieval is less likely to be required for frozen tissue samples.
It is important to have a good idea of expected staining patterns when optimizing an IHC protocol since this will help to determine whether the staining method is performing as it should be. The Human Protein Atlas, an open-access platform that functions to provide detailed mapping of all human proteins within cells, tissues, and organs, is a powerful resource that can guide the selection of suitable positive and negative control tissues.
Image: Immunofluorescence staining of human cell line U-2. Validated by the Human Protein Atlas, anti-AAMP in green localizes to the cytosol and microtubules.
Sensitive and highly specific primary antibodies are essential to the generation of quality IHC data. Furthermore, one should never assume that an antibody that has been validated for staining frozen tissue will also be suitable for an FFPE sample, and vice versa. Antibody manufacturers typically specify which sample type an antibody has been validated against, often providing a recommended staining protocol and representative data.
The choice of primary antibody will also be dictated by the species of the tissue sample. For example, when working with mouse tissue samples it is often recommended to select a primary antibody raised in a species other than mouse to prevent background staining due to recognition of endogenous IgG within the tissue by an anti-mouse secondary antibody. While this can be circumvented using a mouse on mouse blocking reagent, it adds yet another step into an already lengthy protocol.
IHC staining protocols can be shortened considerably using primary antibodies that are directly conjugated to detection moieties, such as enzymes or fluorophores. These also eliminate the requirement to perform secondary-only controls during optimization. Although the range of directly conjugated antibodies is still evolving, it is now possible to perform antibody conjugations in-house using commercially available kits.
Although antibody selection is a critical component of an IHC staining protocol, optimization of concentration, incubation parameters, choice of diluent, and other steps within the procedure should not be underestimated. Additionally, the type of fixative, method of antigen retrieval, blocking, and washing steps all require optimization, while the need to block any endogenous enzyme activity should also be considered. It is also necessary to select an appropriate detection method, for example incorporating streptavidin-HRP to amplify the signal from a biotinylated antibody.
Researchers increasingly demand a greater number of readouts from precious sample material to fully contextualize data. Fluorescent detection provides significant capacity for multiplexing, while the incorporation of mass spectrometry into staining protocols can facilitate antigen quantitation. It is also becoming more commonplace to study mRNA expression levels. Keeping up-to-date on new techniques can greatly increase the quality of information derived from an IHC experiment.
MilliporeSigma offers high-quality reagents for IHC, including over 40,000 Prestige Antibodies® that are supported by the Human Protein Atlas. More information can be found here.