Blocking Non-Specific Binding

Antibody-based applications rely on the specific binding of antibody to the target epitope to generate accurate expression data. Blocking reactive epitopes and endogenous enzymes prior to primary antibody incubation prevents non-specific binding and mitigates false positive error (incorrect identification of positive signal).

How do I block non-specific binding in my immunohistochemistry experiment? Common buffers to block non-specific interactions are normal serum or protein solutions, such as BSA. If blocking with normal serum, the species of the animal serum is dependent on the host of the secondary antibody. For example, use goat serum if using a goat anti-mouse secondary. However, the choice of blocking buffer is contingent on the antigen type and type of detection used. For instance, if an alkaline-phosphatase conjugated secondary antibody is used, then the blocking serum should be diluted in TBS. PBS will interfere with the alkaline phosphatase.

How do I determine non-specific signal in my immunohistochemistry experiment? A negative tissue control (tissue or cells known to lack expression of your target antigen) should be run with each immunohistochemistry experiment to determine the amount of background staining. The presence of non-specific staining will be associated with the deposition of chromogenic or fluorescent signal in the negative tissue control sample.

Blocking Non-specific Binding with Serum. A. CD14 was detected in paraffin-embedded human tonsil tissue using anti-human CD14 biotinylated affinity-purified polyclonal antibody (Catalog # BAF383). Tissue was subjected to antigen retrieval and stained using high sensitivity streptavidin conjugated to HRP (HSS-HRP) and DAB, and counterstained with hematoxylin (blue). B. Non-specific background staining is markedly reduced in a parallel experiment which included a blocking step using animal serum for 15 minutes at room temperature prior to incubation with the primary antibody. (Data provided by R&D Systems)

Blocking Endogenous Peroxidase

What tissues express endogenous peroxidase? Liver, kidney, and other highly vascularized tissues express high amounts of endogenous peroxidase activity.

How does endogenous peroxidase affect my immunohistochemistry results? The presence of endogenous peroxidase can react with HRP conjugated secondary antibodies used in chromogenic IHC staining, resulting in higher non-specific staining and background levels.

How do I block endogenous peroxidase? Treating your tissue with 3-10% hydrogen peroxide prior to incubation with HRP conjugated secondary antibody can quench endogenous peroxidase and significantly reduce non-specific background.

How do I know if my samples express endogenous peroxidase? To determine if your tissue sample expresses endogenous peroxidase, incubate the tissue with DAB substrate after deparaffinization and rehydration. The presence of endogenous peroxidase will be associated with the deposition of brown color.

Quenching Endogenous Peroxidase Activity. A. Failing to quench endogenous peroxidase prior to staining produced a false positive signal in sections of human kidney. Tissue was stained using the anti-goat HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS008; brown). B. Endogenous peroxidase activity was quenched in the same tissue using 3% H₂O₂ in methanol for 15 minutes at room temperature prior to staining.

Blocking Endogenous Biotin

What tissues express endogenous biotin? Liver, kidney, heart, brain, and lung express high amounts of endogenous biotin.

How does endogenous biotin affect my immunohistochemistry results? The presence of endogenous biotin in tissue can increase non-specific staining in chromogenic IHC staining when a biotinylated secondary antibody is used (ABC or LSAB method).

How do I block endogenous biotin? Pre-treating cells with avidin/biotin blocking reagents prior to secondary antibody incubation will reduce non-specific staining due to endogenous biotin. Avidin is routinely used to block endogenous biotin. However, this must be followed by incubation with biotin to block additional biotin binding sites on the avidin molecule.

How do I know if my samples express endogenous biotin? To determine if your samples express endogenous biotin, incubate your tissue section or sample with streptavidin-HRP then DAB, or incubate with ABC complex alone. The presence of endogenous biotin will be associated with the deposition of brown color.

Blocking Optimization: Blocking to Increase Signal:Noise Ratio

Reducing non-specific interactions without limiting antibody binding to the target epitope often requires optimization. Serum and tissue proteins, as well as endogenous molecules (biotin, peroxidase) in tissue samples, can interfere with antibody/target binding and target detection. To determine the optimal signal to noise ratio, empirical testing of the blocking step is recommended. A high ratio of signal to noise indicates a well optimized assay. It is obtained when the signal of the analyte-containing sample (positive sample) is significantly higher than the signal of the analyte non-containing sample (negative sample). Insufficient blocking can result in high background staining, while excessive blocking can result in low signal due to interference between antibody/antigen binding. An optimized blocking step improves signal strength.