Immunostaining: detection of signaling protein location in tissues, cells and subcellular compartments

Abstract

The purpose of this protocol is to describe various methodologies used to detect the distribution and localization of specific proteins within individual cells or tissues using immunostaining, defined as the use of specific antibodies to detect a single target protein. Detection of antigens in cultured cells is referred to as immunocytochemistry, whereas their detection in tissues is generally referred to as immunohistochemistry. Both methods involve exposure of fixed cells or tissues to primary antibodies directed against one or more proteins of interest. Bound antibodies are then detected using commercially available secondary antibodies directed against the invariant portion of the primary antibody. Two primary methodologies exist to visualize antigen-antibody complexes: immunofluorescence using fluorophore-conjugated antibodies or chemiluminescence using antibodies coupled to horse-radish peroxidase. This protocol details the steps involved and appropriate use of both methodologies. Immunostaining is used in cell biology to study differential protein expression, localization and distribution at the tissue, cellular, and subcellular level.