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(If, by leaving out the primary antibody, it has been determined that the secondary antibody is the cause of background)
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.
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 anti-mouse IgG that has been adsorbed against human, for example Alexa Fluor® 488-goat anti-mouse IgG (H+L) (min X Hu, Bov, Hrs Sr Prot), code number 115-545-062, 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 an 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.
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.
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.
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.