Blocking Strategies for IHC
In principle, any protein that does not have binding affinity for the target or probe components in the assay can be used for blocking. In practice, however, certain proteins perform better than others, because they more readily bind to the nonspecific sites (also called reactive sites) or stabilize the function of other system components. In fact, 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 substrate system.
General Blocking Procedures
The blocking step for IHC is most often performed after all other sample preparation is completed and just prior to incubating the sample with the primary antibody. The general protocol is to incubate the fixed, embedded, mounted, cleared and unmasked IHC sample with the appropriate blocking buffer for a time period from 30 minutes to overnight at either ambient temperature or 4ºC based on the optimized protocol specific to each antibody and target antigen. Sufficient washing after the blocking step is critical to remove excess protein that may prevent detection of the target antigen.
Types of Blocking Buffers
Normal serum is a common blocking reagent, because the serum carries antibodies that bind to reactive sites and thus prevents the nonspecific binding of the secondary antibodies used in the assay. A critical factor, though, is to use serum from the species that the secondary antibody was generated in, as opposed to the species of the primary antibody. Serum from the primary antibody species would bind to reactive sites, but the secondary antibody would recognize those nonspecifically-bound antibodies along with the antibodies bound to the target antigen.
Besides serum, concentrated protein buffers made with 0.1 to 5% bovine serum albumin (BSA), gelatin or nonfat dry milk are often used to coat all proteins in a sample. This approach essentially forces primary antibodies to out-compete the blocking protein for binding to cognate ligands while reducing nonspecific binding because the antibodies have no greater binding affinity for nonspecific epitopes than do the buffer proteins. While these buffers can be easily made in the lab, for best results they must be made fresh prior to use, which increases the time and workload of the IHC staining.
Pre-formulated Commercial Buffers
Ready-made blocking buffers are also available to block samples in preparation for antibody treatment. These buffers can contain highly-purified concentrations of 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 or casein and have improved an shelf life.
Guide to Thermo Scientific Pierce Blocking Buffers.
|StartingBlock Blocking Buffers||Single purified protein; fast blocking; broad applicability; excellent for stripping and reprobing Western blots; available in PBS and TBS with and without T20|
|SuperBlock Blocking Buffers||Single purified glycoprotein; fast blocking; broad applicability; stabilizes plate-coated antibodies for drying; available in PBS and TBS with and without T20|
|Blocker BSA Blocking Buffers||Purified bovine serum albumin in PBS or TBS|
|Blocker Casein Blocking Buffers||Purified casein in PBS or TBS|
|Blocker BLOTTO Blocking Buffer||Non-fat dry milk proteins in TBS|
|Pierce Clear Milk Blocking Buffer||Milk proteins, clarified and stabilized in proprietary solution|
|Pierce Fast Blocking Buffer||Proprietary proteins formulation in TBS; developed especially for rapid, 5-minute blocking of Western blots as part of the Fast Western Blot system|
|SEA BLOCK Blocking Buffer||Steelhead salmon serum|
|Protein-Free Blocking Buffers||Proprietary non-protein blocking compound; available in PBS and TBS with and without T20|
Optimizing the Blocking Step
Reducing Background and Increasing Signal:Noise Ratio
By definition, the blocking buffer should improve the sensitivity of the assay by reducing background interference and improving the signal:noise ratio. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether without altering or obscuring the epitope for antibody binding. The proper choice of blocker for a given blot depends on the antigen itself and on the type of detection label used. For example, in applications where alkaline phosphatase conjugates are used, a blocking buffer in TBS should be selected because PBS interferes with alkaline phosphatase.
Empirical testing is essential for true optimization of the blocking step for a particular immunoassay. Many factors, including various protein:protein interactions unique to a given set of immunoassay reagents, can influence nonspecific binding. The most important parameter when selecting a blocker is the signal:noise ratio, measured as the signal obtained with a sample containing the target analyte compared to that obtained with a sample without the target analyte. Inadequate sample blocking will result in excessive background staining and a reduced signal:noise ratio, while excessive blocking may mask antibody:antigen interactions or inhibit the reporter enzyme, again causing a reduction of the signal:noise ratio. When investigating a new target antigen or using a new antibody, it is important to test several different blockers for the highest signal:noise ratio in the assay. No single blocking agent is ideal for every occasion since each antibody-antigen pair has unique characteristics.
- Monitor both background (negative control) and signal strength (positive control) with various blocking reagents
- Choose the blocking buffer that yields the highest signal: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 an Avidin-Biotin Complex system
- For optimal assay conditions, use the same blocking buffer for diluting the antibody that is used for the blocking step
When developing an immunoassay and choosing a blocking buffer, the following trial order is recommended:
- StartingBlock Blocking Buffer
- SuperBlock Blocking Buffer
- SEA BLOCK Blocking Buffer
For Research Use Only. Not for use in diagnostic procedures.