Multiple antigen immunostaining procedures.

Detection of multiple antigens in the same tissue section can be done by combining a range of immunohisto/cytochemical techniques based either on light microscopic chromogenic precipitates or fluorochrome labeling. Light microscopic techniques preferred for this purpose use combinations of immunogold silver staining (black precipitate), immunoperoxidase, immunoalkaline phosphatase and immunogalactosidase methods using chromogens of different colors. Fluorochrome labels favored for these combinations include AMCA (blue), FITC (green), rhodamine (orange-red) and Cy5 (far red), their matching synthetic members from the Alexa series, or quantum dots. Antibodies directly labeled or those from noncross-reacting animal species (e.g., mouse, rabbit, goat, guinea pig etc.) can be applied simultaneously. When the antigens of interest are in separate cells or cell compartments (e.g., in cell membrane, cytoplasm or nucleus), and only cross-reacting antibodies are available, there have also been ways of avoiding unwanted cross-talk. These include the exploitation of the shielding effect of chromogens; inactivation of immuno-sequences of the first staining by using either acidic elution, formaldehyde fixation or microwave heating; combining unlabeled and hapten-labeled antibodies; or using labeled monovalent F(ab) secondary antibodies. In this chapter we briefly discuss the principle of multiple antigen immunolabeling and provide useful protocols for its performance.