Staining with secondary antibody alone was negative (data not shown).

Incubation with primary and secondary antibodies was in the same buffer for 45 min each.

Specific secondary antibodies are usually chosen to work in specific laboratory applications.

Frequently, any one of several secondary antibodies perform adequately in a particular application.

Identifying the optimal secondary antibody is normally done through trial and error.

The secondary antibodies used were cy3-conjugated anti-rat or mouse antibody at 1:200.

This indirect approach permits mass production of secondary antibody that can be bought off the shelf.

Incubations with secondary antibodies were done at room temperature for two hours.

All these antibodies may therefore be recognized by a single secondary antibody.

The secondary antibody, specific to the primary antibody, is added.