Improper pre-blocking of the tissues or cells     top of page

As a general rule, it is safer to pre-block the tissue with 5% normal serum from the same host species as the labeled antibody. The IgG in the serum should occupy sticky sites on the tissue or cells to prevent non-specific binding of the labeled IgG antibody.

Never block the tissue 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 recognised 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. One of the most commonly used protein blockers is bovine serum albumin (BSA). Most of the commercially available BSA is contaminated with bovine IgG (even some of the higher purity grades), which might cause problems when a goat primary antibody is used. The labeled secondary anti-goat will cross-react with bovine IgG significantly since goat and bovine are somewhat closely related.

Cross-reactivity of the labeled secondary antibodies with the endogenous immunoglobulins on the tissues or cells     top of page

Try to look for a labeled secondary antibody that has been adsorbed against the tissue species. 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 so that the anti-mouse IgG will not cross-react with the endogenous human IgG.

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.

Inadequate washing     top of page

This may be a problem when too many slides are jammed 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 come out.

Reactivity of the labeled secondary antibody with the immunoglobulins in the diluent     top of page

For example, if a labeled anti-goat is diluted in diluent containing IgG-contaminated BSA, sticky immune complexes might form due to the cross-reaction of anti-goat with bovine IgG. It is usually not necessary to dilute a labeled secondary in any protein-containing diluent for a few hours of incubation. Therefore, it is safer not to add any protein to the antibody diluent to avoid this type of problem. For a more effective blocking it is better to pre-incubate the tissue with the blocker instead of adding blocker to the antibody diluent. A blocker containing an IgG which cross-reacts with the secondary antibody should not be used even for preblocking.

Not diluting the secondary antibodies far enough     top of page

If background persists after taking all of the above precautions, titrate the labeled secondary antibody further to obtain a maximal signal to noise ratio. Most of the time the labeled secondary antibodies may be diluted further than expected.