Immunohistochemistry (IHC) is a well-known and routinely used technique for the detection and localization of specific proteins, antigens and biomarkers of interest in tissue sections. Results from IHC experiments provide an in-depth study of tissue anatomy and help visualize the expression, localization, and intensity of a specific biomarker. An essential requirement for validating any scientific research finding is the use of appropriate positive and negative controls in the assays and IHC is no exception. The need for suitable controls is a key requirement for robust data interpretation. The quality of the results relies on the analyte, reagents, precise experimental technique and well-designed controls. All these variables account for accurate interpretation of the data.

Six established IHC controls commonly used to determine the specificity of the observed antibody staining.

Positive Controls

Positive controls are specimens containing the biomarker of interest in its known location, whose histomorphology and cytomorphology can be visualized by fluorescent or chromogenic stain. Specific characteristics of the biomarker are initially deduced from other experimental evidence, such as western blots, overexpression, knock-down experiments or studies carried out using experimental models such as transgenic mice, cells and other organisms known to express the biomarker of interest. Conversely, the antibody should not produce any staining in experimental models where the biomarker has been successfully deleted. The use of cell lines transfected to over-express a target, are not recommended as appropriate positive controls, as many assays lack the appropriate dynamic range and calibration to ensure appropriate comparison between a cell line and tissue. The most rigorous positive control is the positive anatomical control; i.e., where the presence of the biomarker in the specimen is known and is not the target of the experimental treatment. For example, an assay that uses an antibody specific for insulin should include sections of pancreas that have islets of Langerhans. The antibody should target only the insulin-producing islet beta cells and not cross-react with closely related molecules of the insulin-like peptide family. To identify a suitable positive control, a good starting place is to check the datasheet. Enzo Life Sciences provides the Uniprot ID on the datasheet so that researchers can access a list of tissues that express the protein of interest, which can be used as positive controls.

Negative Controls

The goal of a negative control is to reveal non-specific binding and false positive results. Negative controls demonstrate that results observed are due to the interaction of the epitope of the target molecule with the antibody. Although the specificity of an antibody can be demonstrated by western blot analysis, the antibody can sometimes provide different results between western blot and IHC applications. The antibody may stain structurally related target molecule by IHC non-specifically in intact cellular and tissue specimens in contrast to samples use for western blot. Common negative controls are tissue samples that do not express the biomarker or protein of interest.

Endogenous Tissue Background Control

Certain cells and tissue types, in particular those abundant in collagen, elastin, and lipofuscin, possess inherent biological properties that emit natural fluorescence. Before applying primary antibodies, cells and tissues should be inspected under the microscope using either fluorescence or bright-field illumination to ensure there is no signal inherent to the tissue itself to rule out endogenous background. If this is unnoticed, it could be misinterpreted as positive staining when it is actually background staining.

No Primary Antibody Control

This is a control in which the tissue is incubated with just the antibody diluent, without the primary antibody. This is necessary to determine if the secondary antibody is binding non-specifically to cellular components that do not contain the biomarker or protein of interest, resulting in false positives or non-specific binding. The samples are incubated with only the antibody diluent without adding the primary antibody. This is followed by incubation and addition of detection reagents. Staining with detection reagents alone should be negligible to the point that it does not obscure specific staining or resemble the specific staining pattern of the biomarker.

Isotype Controls

When working with monoclonal antibodies, including isotype controls in the IHC experiment will validate that, the observed staining is indeed specific to the protein of interest and not produced by non-specific interactions of the antibody with the other components of the tissue samples. An isotype control is an antibody of the same isotype (e.g. IgG2, IgM, IgY), clonality, conjugate, and host species as the primary antibody, which targets a protein or biomaker that is not present in the sample. Instead of incubating the sample with the specific primary antibody, the isotype control sample is incubated with the isotype control antibody at the same concentration under the same experimental conditions.

Absorption Control

To demonstrate that an antibody is binding specifically to the protein of interest, it is first pre-incubated with the immunogen. This should inactivate the antibody and the tissue should show little or no staining. Although highly recommended this type of control is not routinely performed due to limitations in obtaining purified antigens/immunogens.

Enzo provides over 40 years of experience in the manufacturing and supply of research kits, biochemicals, and biologicals. Our growing list of more than 1000 IHC validated antibodies includes those for the detection of key signaling proteins, cell surface markers, and mediators of cell death, oxidative stress, heat shock proteins, proteasomes, and more. We know IHC optimization can be a time and resource consuming endeavor. We back all of our antibodies with our Worry-Free Antibody Trial Program. As Scientists Enabling Healthcare, we strive to provide relevant information to our customers working in the fields of life sciences, drug development and clinical research. We are happy to provide simple and useful tips for your research needs. Please check out our IHC Troubleshooting Guide, 10 Tips for Successful Immunohistochemistry and our Technical Support is ready to offer further assistance.

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