Blocking Strategies for IHC

Before using specific antibodies to detect antigens by immunohistochemistry (IHC), all potential nonspecific binding sites in the tissue sample must be blocked to prevent nonspecific antibody binding. If blocking is omitted or inadequate, the antibodies or other detection reagents may bind to a variety of sites that are not related to specific antibody–antigen reactivity. For example, antibodies can bind to many types of surfaces by simple adsorption, and they can bind to proteins because of charge-based, hydrophobic, and other types of interactions.

Explore IHC Products  Overview of Immunohistochemistry

Page contents

• Introduction
• Types of blocking buffers
• Blocking tips

In principle, any protein that does not bind specifically to the target antigen or the antibodies and other detection reagents in the assay can be used for blocking. In practice, however, certain proteins perform better than others, because they bind readily to nonspecific sites (also called reactive sites) at neutral pH or stabilize the function of other assay components. However, no single protein or mixture of proteins works best for all IHC experiments, and empirical testing is critical to obtain the best possible results for a given combination of specific antibodies and other detection reagents. In the example below, Thermo Scientific Blocker BSA blocking buffer was used in a fluorescent IHC experiment to detect vimentin and lamin B1 proteins.

Fluorescent IHC detection of vimentin and lamin B1 in normal colon tissue. Normal human colon tissue sections were blocked with Blocker BSA blocking buffer (Cat. #37520, 37525) prior to incubation with a mouse anti-vimentin (e.g., Cat. #MA5-14564) and rabbit anti–lamin B1 (e.g., Cat.& #MA1-06103) primary antibodies. The secondary antibodies used were Invitrogen DyLight 405–labeled goat anti-mouse IgG (Cat. #35501BID, blue signal) or DyLight 550–labeled goat anti-rabbit IgG (Cat. #84541, pinkish orange signal), respectively. Cellular nuclei were counterstained green (e.g., Cat. #S7572)

The blocking step for IHC is most often performed after all other sample preparation steps are completed, but just prior to incubating the sample with the primary antibody. The general protocol is to incubate the fixed, embedded, mounted, sectioned, de-paraffinized, and unmasked IHC sample with the appropriate blocking buffer for 30 minutes to overnight at either ambient temperature or 4°C based on an optimized protocol specific for each antibody and target antigen. Sufficient washing after the blocking step is usually performed in order to remove excess protein that may prevent detection of the target antigen. However, many researchers do not wash after the blocking step because they dilute their primary antibodies in their blocking buffer.

Learn more: IHC Troubleshooting Guide

Normal serum at 1-5% (w/v) is a common blocking buffer component, because serum carries antibodies that bind to reactive sites and prevent the nonspecific binding of the secondary antibodies used in the assay. A critical factor, though, is to use serum from the source species for the secondary antibody as opposed to the source species for the primary antibody. This is because serum from the primary antibody species would bind to reactive sites, but the secondary antibody would recognize those nonspecifically-bound antibodies along with the specific antibodies bound to the target antigen. Additionally, serum is rich in albumin and other proteins that readily bind to nonspecific protein-binding sites within the sample.

Besides serum, blocking buffers often contain proteins such as bovine serum albumin (BSA), gelatin or nonfat dry milk added at 1-5% (w/v) final concentrations. These inexpensive and readily available proteins either alone or together are present in large excess compared to the antibody concentration, so they compete with the latter for binding to nonspecific sites in the sample. Many labs have a favorite homemade blocking buffer recipe. However, it is important to make sure that such blocking buffers are free of precipitates and other contaminants that can interfere with IHC detection.

Ready-made blocking buffers are also available to block samples in preparation for antibody treatment. These buffers can contain highly purified single proteins or proprietary protein-free compounds. A benefit of using commercial blockers is that there are many available options that perform better than gelatin, casein or other proteins used alone, and they have improved shelf lives compared to homemade preparations.

We offer several important tips for your blocking experiments:

• Monitor both background (negative control) and signal strength (positive control) with various blocking reagents.
• Choose the blocking buffer that yields the highest signal-to-noise ratio.
• Ensure that there are no substances in the blocking buffer that interfere with a particular assay. Non-fat dry milk, for example, contains biotin and is inappropriate for use with any detection system that includes a biotin-binding protein.
• For optimal assay conditions, use the same blocking buffer for diluting the antibody that is used for the blocking step.

1. Dakshinamurti K, Mistry SP (1963) J Biol Chem 238:294.