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Immunohistochemistry (IHC) experiments often contain one or more blocking steps to reduce background signal and false positives.
These steps include
Blocking with sera or a protein blocking reagent prevents non-specific binding of antibodies to tissue or to Fc receptors. Theoretically, any protein that does not bind to the target antigen can be used for blocking. In practice, some proteins bind more readily to non-specific sites.
Serum is a common blocking agent as it contains antibodies that bind to non-specific sites. Using a serum matching the species of the secondary antibody is recommended. When performing multiple stains using secondary antibodies from different species, it may be necessary to use blocking sera from the species of both secondary antibodies.
Proteins such as BSA or casein may also be used to block non-specific antibody binding, and with these there is no need to match the reagent to the species of the secondary antibody.
Specialized blocking buffers are also frequently used to block non-specific antibody binding. These are often formulated to optimize performance and/or shelf life.
When staining mouse tissues with mouse primary antibodies, there is often high background 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 this protocol. It is often important to carefully match the blocking fragment and the secondary antibody used.
If a biotin-based detection system is used, blocking endogenous biotin is recommended. Biotin is present in many tissues, particularly in kidney, liver and brain. It is blocked by pre-incubation of the tissue with avidin, followed by incubation with biotin to block additional biotin binding sites on the avidin molecule.
Chromogenic detection methods usually use an enzyme linked directly, or indirectly, to a secondary antibody to visualize antibody localization. If the enzyme is naturally present in the tissue being studied, its activity must be blocked before the detection step.
When using HRP for detection, non-specific or high background staining may occur due to endogenous peroxidase activity. Tissues such as kidney, liver and those containing red blood cells (such as vascular tissue) contain endogenous peroxidases. To check for endogenous peroxidase activity, tissues can be incubated with DAB substrate prior to primary antibody incubation. If tissues turn 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.
Alkaline phosphatase blocking
Endogenous alkaline phosphatase (AP) can produce a high background when using AP for detection. It can be found in kidney, intestine, osteoblasts, lymphoid tissue and placenta. AP activity is higher in frozen tissue. Tissue can be tested for endogenous AP by incubating with BCIP/NBT; if a blue color is observed, endogenous AP is present and blocking is necessary. Levamisole is used for blocking, and is added with the chromogenic substrate. Intestinal AP is blocked with a weak acid (eg 1% acetic acid) before adding the primary antibody.
When using a fluorescent label for detection, there is a possibility that the tissue may be autofluorescent, leading to high background. Tissue fixation may induce autofluorescence, particularly when using aldehyde fixatives (eg formalin), which react with amines to generate fluorescent products.
Autofluorescence may also be caused by the presence of fluorescent compounds, such as flavins and porphyrins. These compounds may be extracted 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.
To reduce autofluorescence, either use non-aldehyde fixatives, such as Carnoy’s solution, or block aldehydes by treating with sodium borohydride or glycine/lysine. Alternatively, try using frozen tissue sections, or treating tissue with quenching dyes such as pontamine sky blue, Sudan black, trypan blue or FITC block,
If endogenous autofluorescence cannot be blocked, a chromogenic detection system may be preferable.
View FITC Protein Blocking Agent used to reduce autofluorescence.