Blocking (http://www.piercenet.

com/method/blocking-strategies-ihc) In principle, any protein that does not have binding affinity for the target or probe components in the assay can be used for blocking. In practice, however, certain proteins perform better than others, because they more readily bind to the nonspecific sites (also called reactive sites) or stabilize the function of other system components. In fact, no single protein or mixture of proteins works best for all IHC experiments, and empirical testing is critical to obtain the best possible results for a given combination of specific antibodies and substrate system.

General Blocking Procedures The blocking step for IHC is most often performed after all other sample preparation is completed and just prior to incubating the sample with the primary antibody. The general protocol is to incubate the fixed, embedded, mounted, cleared and unmasked IHC sample with the appropriate blocking buffer for a time period from 30 minutes to overnight at either ambient temperature or 4C based on the optimized protocol specific to each antibody and target antigen. Sufficient washing after the blocking step is critical to remove excess protein that may prevent detection of the target antigen.

Types of Blocking Buffers

Normal Serum Normal serum is a common blocking reagent, because the serum carries antibodies that bind to reactive sites and thus prevents the nonspecific binding of the secondary antibodies used in the assay. A critical factor, though, is to use serum from the species that the secondary antibody was generated in, as opposed to the species of the primary antibody. Serum from the primary antibody species would bind to reactive sites, but the secondary antibody would recognize those nonspecifically-bound antibodies along with the antibodies bound to the target antigen. Protein Solutions Besides serum, concentrated protein buffers made with 0.1 to 5% bovine serum albumin (BSA), gelatin or nonfat dry milk are often used to coat all proteins in a sample. This approach essentially forces primary antibodies to out-compete the blocking protein for binding to cognate ligands while reducing nonspecific binding because the antibodies have no greater binding affinity for nonspecific epitopes than do the buffer proteins. While these buffers can be easily made in the lab, for best results they must be made fresh prior to use, which increases the time and workload of the IHC staining. Pre-formulated Commercial Buffers

Ready-made blocking buffers are also available to block samples in preparation for antibody treatment. These buffers can contain highly-purified concentrations of single proteins or proprietary protein-free compounds. A benefit of using commercial blockers is that there are many available options that perform better than gelatin or casein and have improved an shelf life.
http://answers.yahoo.com/question/index?qid=20080130051920AAW0oYH

Blocking is done to prevent non-specific binding of either your primary or secondary antibody. You add an excess of blocking agent to saturate (block) any nonspecific binding sites that your Ab's might otherwise stick to. Then, when you add your primary Ab, it only sees the specific binding sites (i.e. the antigen that it recognizes). Since the blocking step is for non-specific binding, you don't need a separate blocking step for primary & secondary antibodies. You just need one blocking step that works well. Using normal serum makes sense, because it will contain a mixture of antibodies. They won't specifically recognize your target antigen, since they come from a nonimmunized animal. However, they will stick to anything that tends to bind Ab's nonspecifically. Why use serum from the secondary host instead of the primary host? Imagine you have a tissue that you want to stain for Protein X, using IHC. First you block, then you incubate with primary Ab. Let's assume that's a rabbit antibody against Protein X. Next you add your secondary antibody. That might be something like fluoresceinlabeled goat anti-rabbit antibody. In other words, it's an antibody from a goat, and it specifically binds to antibodies from rabbits. Any place where your primary rabbit antibody has bound to Protein X, the goat secondary antibody will bind to it. Since the secondary is labeled (with fluorescein, in this example), you can detect it. Now go back to the blocking step. If you used goat serum to block, any nonspecific binding sites have random goat antibodies stuck to them. THat's find, because the goal secondary Ab won't recognize any of those. But imagine if you'd used rabbit serum to block. All those non-specific sites would have random rabbit antibodies stuck to them. The goat secondary would recognize all those nonspecifically bound rabbit antibodies, as well as any specifically bound primary antibody to Protein X. YOu wouldn't be able to tell the difference between binding to Protein X and non-specific binding. Hope that makes sense and is helpful. Sorry for being long-winded.

Source: Ph.D. in molecular biology

http://www.jacksonimmuno.com/technical/techq1.asp#top

Improper blocking of the tissues or cells

top of page

As a general rule, it is most effective to block tissue or cells with 5% normal serum from the same host species as the labeled secondary or tertiary antibody. The IgG in serum should occupy sticky sites on the tissue or cells to prevent non-specific binding of the labeled IgG antibody. Never block the tissue or cells with normal serum from the same host species as the primary antibody. For example, if a primary antibody is made in mouse and normal mouse serum were used for blocking, the mouse IgG would bind to the sticky sites and be recognized by labeled anti-mouse IgG. A higher background would result. Make sure a universal blocker does not contain any IgG that the labeled secondary antibody might cross-react with. Two of the most commonly used protein blockers are bovine serum albumin (BSA) and dry milk. However, most of the commercially available BSA and dry milk are contaminated with bovine IgG (even some of the higher purity grades of BSA), which might cause problems when a goat or sheep primary antibody is used. The labeled secondary anti-goat will significantly cross-react with bovine IgG since goat and sheep are very closely related to cow. These principles apply as well to all immunological procedures including ELISA, Western blots, flow cytometry, immunohistochemistry, and immunocytochemistry. Cross-reactivity of labeled secondary antibodies with endogenous immunglobulins on the tissues or cells Select a labeled secondary antibody which has been adsorbed against the species of the experimental tissue, if possible. For example, if the tissue is human tonsil and the primary antibody is made in mouse, use a labeled antimouse IgG that has been adsorbed against human, for example DyLight 488-goat anti-mouse IgG (H+L) (min X Hu, Bov, Hrs Sr Prot), code number 115-485-062, so that the anti-mouse IgG will not cross-react with any endogenous human IgG in the tissue. If a mouse tissue that contains immunoglobulins is used and the primary

antibody is also made in mouse, the endogenous immunoglobulins may be blocked with a monovalent Fab fragment of anti-mouse IgG. To avoid subsequent challenge by the labeled anti-mouse IgG, the tissue may be lightly fixed after Fab antibody blocking, provided this treatment does not alter the antigen to be detected. Why use a Fab monovalent antibody? If a divalent antibody is used for blocking, the second binding site on the antibody might be open to capture the subsequent primary antibody and thus amplify the background. Inadequate washing This may be a problem when too many slides are placed in the same washing chamber. This may also be a problem when labeling a whole mount or a thick section that allows poor penetration. If it takes a long time for antibodies to penetrate into the cells, it should also take a long time for the excess antibodies to diffuse out. The washing step may need to be extended to wash out unbound antibody. Reactivity of labeled secondary antibodies with immunoglobulins in the diluents It is usually not necessary to dilute a labeled secondary antibody in any protein-containing diluent. For example, if a labeled anti-goat IgG is diluted in diluent containing IgG-contaminated BSA or dry milk, sticky immune complexes might form due to the cross-reaction of anti-goat IgG with bovine IgG. For more effective blocking, it is better to pre-incubate the tissue with the blocker instead of adding the blocker to the antibody diluent. A blocker containing IgG which cross-reacts with the secondary antibody should not be used. Not diluting the secondary antibodies far enough If background persists after taking all of the above precautions, titrate the labeled secondary antibody further to obtain a maximal signal to noise ratio. Labeled secondary antibodies often may be diluted further than expected.