Antigen (tissue) controls

Positive control

A positive control is known to express the protein of interest. Even if the samples are negative, positive results from the positive control will indicate that the procedure is working and optimized, verifying that any negative results are valid.

Negative control

A negative control is known not to express the target antigen. This is to check for non-specific signals and false-positive results.

Endogenous tissue background control

An endogenous tissue background control is a section from the tissue before applying the primary antibody. Certain tissues have inherent properties that result in background staining, affecting the interpretation of results. For example, tissues containing endogenous fluorescent molecules could be confused for positive staining during fluorescent IHC. The tissue should be checked under the microscope using either fluorescent or bright-field illumination (for fluorescent or chromogenic labels, respectively) to ensure no endogenous background.

Reagent controls

No primary antibody control

A no primary antibody control confirms the staining is produced from detection of the antigen by the primary antibody and not by the detection system or the specimen. This involves incubating the sample with the antibody diluent alone and no primary antibody, followed by incubation with secondary antibodies and detection reagents.

Isotype control

An isotype control can be used when working with monoclonal primary antibodies, and checks that the observed staining is not caused by non-specific interactions of the antibody with the tissue. The tissue is incubated with the antibody diluent and a non-immune antibody of the same isotype at the same concentration as the primary antibody, followed by incubation with secondary antibodies and detection reagents. Any background staining observed with this control should be negligible and distinct from specific staining.

Absorption control

An absorption control demonstrates that the antibody binds specifically to the antigen of interest by incubation with a pre-absorbed antibody instead of the primary antibody, followed by incubation with secondary antibodies and detection reagents. A pre-absorbed antibody may be produced by overnight incubation of the antibody at 4°C with a large molar excess (10-fold) of the immunogen.

Absorption controls are more reliable if the immunogen is a peptide, as antibody-protein immunogen mixtures may themselves cause high background staining in tissues due to non-specific interactions between the protein and the tissue.