The indirect method of IHC staining consists of multiple steps, as shown in the illustration. Indirect staining methods use a secondary labeled antibody with specificity against the unlabeled primary antibody. This secondary antibody is produced against immunoglobulin (IgG) of the species from which the primary antibody has been derived from. Multiple secondary antibodies will bind to the primary antibody, resulting in the amplification of the antigen-antibody reaction. The reaction signal is further enhanced when the secondary antibody is conjugated with a fluorophore (fluorescent chemical compound) or biotin.
The indirect method of IHC staining is significantly more sensitive than the direct method. Because of this, it is the most commonly used method for clinical staining applications.
