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Basics of the Blocking Step in IHC

After you have successfully pre-treated your IHC sample, it may be required to complete a blocking step. Blocking is not always required but may be depending on factors such as the primary or secondary antibody, what kind of detection you’re using, as well as what kind of tissue you have. The main purpose of blocking reagents is to reduce background or non-specific staining, thus reducing the chance for a false positive.


Types of Blocking Reagents


Hydrogen Peroxide: If you are using DAB chromogen with horseradish peroxidase (HRP) as the detection enzyme, the tissue may need to be incubated in hydrogen peroxide solution to block the staining of endogenous peroxidases. In tissues that are high in endogenous peroxidases, such as bloody tissues and tissues high in red blood cells like bone marrow or spleen, the DAB will link to those and stain non-specifically.


Levamisole: Levamisole is the equivalent solution to hydrogen peroxide for blocking endogenous alkaline phosphatase when using alkaline phosphatase (AP) instead of HRP as the detection enzyme. It will block the staining of endogenous alkaline phosphatase in tissues like kidney or lymphoid. Levamisole is ineffective against intestinal alkaline phosphatase however, so in those cases acetic acid is used.


Avidin/Biotin Blocking Reagents: Avidin/Biotin blocking reagents are used to block endogenous biotin, which can be found in tissues such as liver, kidney, heart, lung, or brain. This blocking is usually done when using ABC (Avidin Biotin Complex) or LSAB (Labeled Steptavidin-Biotin) method of detection. Here, biotin is blocked by incubating the tissue in avidin, which bonds easily with biotin, and then incubating again with biotin, to block the remaining biotin binding sites on the avidin that was added.


Protein Blocking Solutions: Protein blocking solutions are applied after pre-treatment to prevent non-specific binding of the detection reagent to cell proteins. Essentially, you’re applying another protein to come in and bind to the non-specific sites (the sites that are not the antigen), so that the detection reagent can’t bind to them. Serum is a common blocking agent since the antibodies it contains will bind to non-specific sites. The species the serum comes from should match the species of the secondary antibody being used. If you use the species from the primary antibody, it will bind to the non-specific sites, but the secondary antibody will recognize both the non-specific bound sites, and the antigen sites.


In addition to regular serum you’ll also find other blocking buffers that contain things like BSA (bovine serum albumin), gelatin or non-fat dry milk. You’ll want to be careful that whatever blocking solution you use doesn’t contain anything that would interfere with your assay. For example, if you’re using a biotin-binding protein, you won’t want to use dry milk because it contains biotin.


To determine if a blocking reagent is needed, you should run a negative tissue control, something known to lack your target antigen, and see if you’re getting chromogen signal. If you are, you will need a blocking reagent to eliminate that background staining.


Interested in learning more about the basics of IHC, check out the 2020 Convention Basics Package, an on-demand webinar package containing 5 webinars from the 2020 NSH Convention that cover basic histology skills, including microtomy troubleshooting, choosing a fixative, tissue recognition, and embedding.


Resources:

https://www.biotrend.com/en/buy/cat-ihc-buffers-quenching-ap-4182.html

https://www.clinisciences.com/en/buy/cat-hrp-chromogen-for-ihc-dab-3862.html

https://www.abcam.com/kits/blocking-for-ihc

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/blocking-strategies-ihc.html

https://www.novusbio.com/blocking-non-specific-binding

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/avidin-biotin-complex-method-ihc-detection.html





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