The page below is a sample from the LabCE course Immunohistochemistry (IHC) Basics in Histology. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Indirect Method

The indirect method consists of using one antibody against a specific antigen and then adding a second, labeled (meaning in IHC an attachment of a substance such as a fluorescent dye, enzyme, or radioactive substance to a protein/antibody for visualization of an antigen) antibody against the first called a secondary antibody.
The technique methodology consists of using an unlabeled primary antibody. Then, a labeled secondary antibody, which must be raised against the IgG of the animal species in which the primary antibody has been raised, is used to react with the primary antibody.
The final step is detection of the antibody/antigen reaction. Depending on what the secondary antibody is labeled with determines how and what detection method is incorporated, such as a fluorescent dye or biotinylation (biotin). There are blocking methods incorporated into this method that help to decrease background staining.