By Stewart Newlove, PhD and Ryan Hamnett, PhD
Immunocytochemistry/Immunofluorescence (ICC-IF) involves using antibodies to selectively label proteins of interest in cells and visualize them with a fluorescent microscope. Controls are essential to ensure the accuracy, specificity, and reliability of the ICC-IF assay. Positive controls ensure the reagents and protocol parameters are sufficient for enabling specific detection of the target antigen, while negative controls assess background staining caused by non-specific interactions. Common approaches to implementing such controls are outlined in the table below.
| Control | Description | Purpose | Notes |
|---|---|---|---|
| Positive expression control | Stain alongside a cell type known to express protein of interest, or overexpress the protein in experimental cell line. | Confirms whether negative staining observed in the experimental sample represents a true negative result, or is a technical error. | Information about a suitable cell line may be available on the antibody’s datasheet, in online databases, or can be found in the literature. |
| Negative expression control | Stain alongside a cell type that does not express protein of interest, either natively or through lack of transfection. | Confirms that the staining in the experimental sample is valid, or if the primary antibody exhibits non-specific staining. | Negative controls include cells from knockout organisms. |
| 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 cell components). |
| Endogenous background control | Visualize unstained sample (fixed and blocked but not immunostained) under microscope. | Detects inherent signal in cells not attributable to antibody staining. | |
| 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 ICC experiment.