Once tissue or cell samples have been appropriately prepared and fixed, the samples are ready to be stained. All IHC/ICC studies are dependent on specific antibody-epitope binding, which is governed by hydrophobic interactions, ionic interactions, hydrogen bonding, and other intermolecular forces. However, the same attractive forces can also result in non-specific staining, i.e. binding of the primary antibody to amino acids other than those within the desired epitope of the antigen. This is a common problem that occurs in IHC/ICC experiments. The challenge is to reduce non-specific interactions without impairing antibody-epitope binding. Causes of non-specific staining include interactions of the primary and secondary antibodies with serum proteins, ionic interactions between antibodies and tissues, and interactions with endogenous molecules capable of affecting the IHC detection system used. These issues can result in high background and an inability to visualize the antigen of interest in its appropriate cellular location. Staining problems of this type can be addressed by blocking non-specific interactions using a blocking reagent. These steps are performed prior to incubation of the sample with the primary antibody.
Although hydrophobic interactions play an essential role in epitope-antibody binding, these forces can also promote non-specific binding. Most proteins have some degree of hydrophobicity due to the neutral side chains of several amino acids. Tissue incubation with heat-inactivated normal serum or bovine serum albumin (BSA) is a common procedure used to reduce non-specific hydrophobic binding. Selection of the type of normal serum is important to prevent interactions with the primary or secondary antibodies, or with the tissues/cells being stained. For example, goat serum would not be advised as a blocking reagent for use with a goat-derived primary antibody. Instead, a serum identical to the host animal of the secondary antibody or from an unrelated species is recommended. BSA and non-fat dry milk are also frequently used as blocking reagents. One of these reagents is typically included in the diluents for the primary and secondary antibodies. The addition of non-ionic detergents including 0.3% Triton X-100™ or Tween 20™ can also reduce non-specific hydrophobic interactions.
|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 using the Basic Antigen Retrieval Kit (Catalog # CTS013). Tissue was 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. HSS-HRP and animal blocking serums are included in all R&D Systems Cell and Tissue Staining Kits.|
If the antibody employed and the target tissue have net opposite charge, ionic interactions can cause non-specific background staining. For example, non-specific staining may be due to attraction between opposing carboxyl and amino groups. Van der Waals forces, weak electrostatic interactions between dipolar molecules, can also contribute. Increasing the ionic strength of the fixative and/or the antibody diluent buffer can reduce ionic interactions. However, epitope-antibody binding is commonly dependent on ionic forces, so this approach also has the potential to negatively impact staining specificity. Due to their single epitope specificity, increasing ionic strength is more likely to impair monoclonal than polyclonal antibody performance.
Chromogenic detection methods often use a conjugated enzyme to visualize epitope-antibody interactions. When using this method of detection, the endogenous activity of the same enzyme must be blocked. For example, protocols that include horseradish peroxidase (HRP) or alkaline phosphatase (AP) may require reagents to prevent non-specific signals. Tissues such as kidney, liver, or vascular areas with red blood cells, contain endogenous peroxidase activity. Peroxidase blocking reagents formulated with 3-10% H2O2 can be used to prevent endogenous peroxidase from cleaving the substrate. Endogenous AP found in intestine, kidney, lymphoid and other tissue can be blocked with 1 mM Levamisole. The intestinal form of AP is unaffected by Levamisole but can be blocked by using 1% acetic acid.
|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.|
Streptavidin binding to a biotinylated primary or secondary antibody is another common detection system that is utilized for IHC/ICC experiments. Therefore endogenous biotin must be blocked before streptavidin incubation. Endogenous biotin is found in many tissues including liver, kidney, heart, brain, and lung. Pre-incubation of the sample with avidin is routinely used to block endogenous biotin. This must be followed by a subsequent incubation with biotin to block additional biotin binding sites on the avidin molecule. Following these biotin/avidin blocking steps, the sample can be incubated with the primary antibody.
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