Multiple Immunofluorescence Labeling of Formalin-Fixed Paraffin-Embedded Tissue

Multiple immunofluorescent labeling of formalin-fixed paraffin-embedded (FFPE) tissue is not a routinely used method. At least in part, this is due to the perception that the innate autofluorescence of the FFPE material forbids the use of immunofluorescent labeling. As a result, immunohistochemical (immunoperoxidase) staining of FFPE material or cryosectioning methods is used instead. In this chapter, we describe a robust optimized method for high-resolution immunofluorescence labeling of FFPE tissue that involves the combination of antigen retrieval, indirect immunofluorescence, and confocal laser scanning microscopy. Once such samples have been prepared and imaged by confocal microscopy, they can be stored at −20�C for extensive periods (>250 days) and reexamined with minimal loss of quality. As a consequence, this method has the potential to open up the large archival sample collections to multiple immunofluorescent investigations.