Tips for Reducing Non-specific Staining in IHC

Immunohistochemistry (IHC) can be a powerful tool for visualizing antigens of interest within the context of cells and tissues. But what can you do when non-specific staining in your IHC is obscuring your results and incurring your PI’s disdain? Consider these factors to help reduce non-specific staining in your IHC experiments.

Fixation

Over-fixation can cause high levels of background in IHC experiments. Try reducing the time your tissue spends in fixative to reduce background. When deciding on a fixation protocol, it’s important to take into account the size of the tissue you are fixing. Whole animal perfusion might be necessary for large tissues whereas smaller dissected tissues can be fixed by immersion in fixative.

Slide Preparation

Decreasing the thickness of tissue sections can help keep reduce background staining. Try cutting thinner sections for cleaner IHC. It’s also important to take care not to let sections dry out since drying can result in high non-specific staining.

Deparaffinization

Incomplete deparaffinization is another common cause of high background in IHC experiments. Using fresh xylene will help ensure complete deparaffinization. Protocols vary in the number of xylene washes as well as wash time. Try an additional xylene wash or increasing wash time if high background continues to be a problem.

Blocking

Blocking non-specific binding sites is critical for preventing non-specific staining. Blocking is typically done with normal serum from the species in which the secondary antibody was raised, although other blocking agents (such as BSA) are sometimes used. Increasing the concentration of blocking agent and incubation time in blocking might help to reduce non-specific staining in your experiment.

Antibodies

Using too much antibody will result in high non-specific staining. Try reducing the concentration of primary antibody. Decreasing incubation time or temperature can also help reduce non-specific staining. Doing a control experiment where no primary antibody is used can be helpful to determine if your secondary antibody is causing non-specific staining. When using a mouse primary antibody and working with mouse tissue, high background can result from secondary antibody binding to endogenous immunoglobulins in the tissue. In this case, it can be helpful to use a different primary antibody or try a pre-absorbed secondary antibody. It is also possible to eliminate the need for secondary antibody by using a conjugated primary. Although this will result in a cleaner stain, some sensitivity will be lost since secondary antibodies amplify signal.

Endogenous Peroxidases and Biotin

When using chromogenic detection, endogenous peroxidases may be to blame for high background. Endogenous peroxidases are able to convert substrate and cause non-specific staining. Testing for peroxidase activity can be performed by taking a rehydrated tissue section and applying substrate. If colored precipitate appears, try adding treatment with hydrogen peroxide (used to quench endogenous peroxidases) before antibody binding to your protocol.

Detection systems that employ avidin-biotin complexes are susceptible to high background caused by endogenous biotin. In order to block endogenous biotin, samples can be treated with an excess of unlabeled streptavidin (to bind endogenous biotin) and then treated with an excess of biotin (to saturate biotin binding sites on the streptavidin). This will result in endogenous biotin being bound to streptavidin and all the biotin binding sites on the streptavidin being filled.

Further reading:

Suppression of Endogenous Avidin-binding Activity in Tissues and Its Relevance to Biotin-Avidin Detection Systems