By Rachel Stewart, PhD and Ryan Hamnett, PhD
Immunohistochemistry (IHC) involves using antibodies to selectively label proteins of interest in tissue samples. Controls are essential for confirming the accuracy of antibody staining, and should be included for the first use of a primary antibody in a target tissue to validate immunostaining results.
| Control | Description | Purpose | Notes |
|---|---|---|---|
| Positive expression control | Stain alongside a tissue known to express protein of interest. | Confirms whether negative staining observed in the experimental sample represents a true negative result, or is a technical error. | Information about a suitable tissue may be available on the antibody’s datasheet, in online databases, or can be found in the literature. |
| Negative expression control | Stain alongside a tissue that does not express protein of interest. | Confirms that the staining in the experimental sample is valid, or if the primary antibody exhibits non-specific staining. | Negative control tissues include samples from knockout organisms or simply another region of the tissue. |
| Secondary antibody control | Sample is incubated with only the secondary antibody, no primary antibody. | Indicates non-specific staining due to the secondary antibody. | |
| Isotype control | Substitute primary antibody for another primary antibody of the same isotype (e.g. IgG1, IgM) whose target is not in the sample, or a non-immune immunoglobulin. | Detects non-specific interactions between immunoglobulin and the sample. | Particularly for when working with monoclonal antibodies. |
| Absorption control | Incubate the primary antibody with the peptide immunogen used to generate the antibody. | Confirms specific binding between the primary antibody and the antigen; pre-absorption should remove genuine signal. | Can give false negatives (if target epitope is similar in different proteins, pre-absorption will prevent binding to all), and false positives (if the bound immunogen non-specifically binds to tissue components). |
| Endogenous background control | Visualize unstained sample (fixed and blocked but not immunostained) under microscope. | Detects inherent signal in tissue not attributable to antibody staining. | Applicable to both fluorescent and chromogenic staining. |
| Endogenous enzyme activity control | Apply enzyme (e.g. HRP or AP) substrate to tissue in the absence of antibodies. | Detects inherent enzyme activity not attributable to enzyme-conjugated antibodies. | This is only necessary when using an enzyme-based amplification step. |
| Specificity control | Test the antibody in an alternative application, such as western blot. | Validates that the antibody is detecting a protein of the expected weight with minimal cross-reactivity (i.e. no or few other bands). | Differences in protein conformation between IHC and WB can render negative results of this control misleading, particularly for monoclonal antibodies that only target one epitope. |
Table 1:Key controls to run alongside an IHC experiment.