Immunohistochemistry (IHC) is a widely used technique that relies on antibody binding to specific antigens within a preserved tissue section. It allows the anatomy of the tissue to be studied, and also provides information regarding antigen localization and expression levels. Although IHC is relatively straightforward to perform, as with any form of immunodetection the quality of the staining can be influenced by several variables. Jonathan Weinreich, an experienced product manager at Enzo Life Sciences, spoke with Biocompare to provide his top tips for successful IHC staining.
“When designing an IHC experiment”, says Weinreich, “you must first decide whether your tissue will be frozen, or formalin-fixed paraffin-embedded (FFPE). Both methods preserve samples well, but the type of downstream analysis you intend to perform will dictate which one you select. While freezing samples is extremely rapid, freezing may lead to a loss of morphological integrity. FFPE samples can conveniently be stored at room temperature, however they are time-consuming to prepare, and the embedding process may cause cell shrinkage, resulting in a deceptively high antigen density over a given tissue area. Whichever method you choose, it’s imperative that the sections are smooth and uniform for accurate interpretation of the data.”
Formalin fixation results in protein cross-links that mask antigens and prevent antibody binding, therefore antigen retrieval is recommended on most FFPE sections. Proteolytic-induced epitope retrieval (PIER) uses enzymes such as proteinase K, trypsin, or pepsin to digest protein cross-links, whereas heat-induced epitope retrieval (HIER) relies on heat. “The antigen retrieval method will require optimization” says Weinreich, “and we usually recommend HIER, unless otherwise stated by the antibody manufacturer, since PIER has been known to alter tissue morphology.” Antigen retrieval is not usually recommended for frozen tissue since the heating process may damage the sample.
Practically all immunostaining protocols involve a blocking step to prevent non-specific antibody binding. “The main source of background staining during IHC occurs due to non-specific primary antibody binding to Fc receptors in the tissue sample, therefore blocking is essential,” says Weinreich. “Researchers typically block with serum from the host species of the secondary antibody, however we recommend our universal Antibody Blocker/Diluent, a superior serum-based blocking solution, which really comes into its own when performing a multiplexing experiment.”
Prior to blocking non-specific antibody binding sites, it’s essential that endogenous biotin, peroxidases, and phosphatases are blocked too. “Biotin occurs naturally in tissue samples,” says Weinreich, “and can produce false positives if the detection system is based on avidin or streptavidin binding. Many tissues also express alkaline phosphatase and peroxidases, which can generate artefacts when using alkaline phosphatase- or HRP-conjugated secondary antibodies. Our universal Enzyme Blocking Reagent suppresses non-specific staining due to endogenous enzyme activity; it’s quick and easy to use, and compatible with both frozen and FFPE sections.”
It’s vital to check the characteristics of the primary antibody before using it to stain a precious tissue sample, and Weinreich highlights the utility of Western blotting for evaluating an antibody’s performance and specificity. “A Western blot is a great way to ensure that a primary antibody is suitable for IHC” says Weinreich, “and is easily performed using positive and negative cell lysates. You can next optimize the working concentration of the antibody by using it to stain positive and negative cells embedded in paraffin.” Appropriate controls should always be included to allow correct interpretation of data. “Online databases such as GeneCards, Human Protein Atlas, or Uniprot should help you identify the appropriate positive controls for your experiment” adds Weinreich, “while as negative controls we recommend knock-out or knock-down tissue samples. You should also include a control with no primary antibody, and an isotype control.”
The choice of detection system will be dictated by the expression levels of the target of interest, with conjugated primary antibodies often preferred for antigens of high abundance, and labeled secondary antibodies favored for low to medium-abundance antigens by virtue of the signal amplification they provide. Although avidin-biotin and streptavidin-biotin detection systems are most commonly used for IHC, enzyme-conjugated polymer reagents are becoming increasingly popular. “Not only do these reagents significantly reduce the amount of primary antibody that’s required,” says Weinreich, “they also circumvent endogenous biotin background and produce really sharp staining.”
By thoroughly researching the target of interest, and incorporating all necessary steps into the staining protocol, IHC can provide highly valuable information. Enzo Life Sciences offers a wealth of experience, along with the high-quality reagents to ensure successful IHC. For more information, visit enzolifesciences.com/IHC.