Blocking in IHC

Serum is a common protein-blocking agent as it contains antibodies that bind to non-specific sites. For best results, use a serum that matches the secondary antibody species; this prevents the detection of non-specific binding sites (Figure 2). If you are performing multiple stains using secondary antibodies from different species, it may be necessary to use blocking sera from the species of both secondary antibodies.

Other commonly used protein-blocking reagents are bovine serum albumin (BSA) and casein from nonfat dry milk. Pre-formulated blocking buffers are also available, which are often formulated to optimize performance or shelf life. These methods do not need to be matched to the species of the secondary antibody.

Extra care must be taken when using mouse primary antibodies with mouse tissue. High background staining can occur as the anti-mouse secondary antibody binds to endogenous mouse IgG and Fc receptors. This background binding can be reduced using F(ab) fragments, as described in our mouse-on-mouse protocol, and it is important to match the blocking fragment carefully and the secondary antibody used.

If a fluorescent label is used for detection, check if your tissue is autofluorescent, as this can lead to false-positive results and high background. There are several causes of autofluorescence, including the fixation method and fluorescent compounds. However, if endogenous ​autofluorescence cannot be blocked, a chromogenic detection system may be preferable.

Causes and tips to reduce autofluorescence:

• Tissue fixation, particularly when using aldehyde fixatives (eg formalin), which react with amines to generate fluorescent products.This can be reduced through:

  • Use non-aldehyde fixatives, such as Carnoy’s solution

  • Block aldehydes by treating samples with sodium borohydride or glycine/lysine.

  • Use frozen tissue sections

  • Treat tissue with quenching dyes such as pontamine sky blue, Sudan black, trypan blue, or FITC block.

• Fluorescent compounds, such as flavins and porphyrins. The effect of these can be reduced through: 

  • Extract these compounds from the tissue by the solvents used to generate fixed, dehydrated sections. However, they persist in frozen sections that have been processed using aqueous reagents.

Chromogenic detection uses enzymes to visualize the target antigen. Therefore, endogenous enzymes need to be blocked before detection.

If you are using a biotin-based detection system, blocking endogenous biotin is recommended. First, an excess of avidin is added to the sample to bind any naturally occurring biotin. This is followed by incubation with an excess of biotin to block additional binding sites on the avidin molecule.

Consider using a polymer-based method if the sample is high in endogenous biotin, such as the kidney, liver, and brain. Polymer methods use a dextran backbone to which multiple enzyme molecules and secondary antibodies are attached.

Some tissues, including the kidney, liver, and those containing red blood cells (such as vascular tissue), contain endogenous peroxidases. If you are using horseradish peroxidase (HRP) to view proteins of interest, check for endogenous peroxidase activity by incubating the sample in 3,3'-diaminobenzidine (DAB) substrate. If the sample turns brown, endogenous peroxidase is present, and a blocking step is required. A 10-15 minute incubation in 0.3% hydrogen peroxide is usually sufficient blocking.

​Endogenous alkaline phosphatase (AP) can be found in the kidney, intestine, osteoblasts, lymphoid tissue, and placenta and generally has higher activity in frozen tissue.   

If you are using AP for detection, determine if the sample contains endogenous AP by incubation with a BCIP/NBT solution. If the sample turns blue, endogenous AP is present, and blocking is necessary. Blocking is achieved by incubation with an alkaline phosphatase inhibitor, such as levamisole hydrochloride or tetramisole hydrochloride.